Human hematopoietic stem and progenitor cell antigen and methods for its use

ABSTRACT

A hematopoietic progenitor cell antigen and reagents, notably antibodies, that specifically bind to the antigen are provided. Expression of the antigen is highly tissue specific. It is only detected on a subset of hematopoietic progenitor cells derived from human bone marrow, fetal bone marrow and liver, cord blood and adult peripheral blood. The subset of cells recognized by AC133 is CD34 bright  and contains substantially all of the CFU-GM activity present in the CD34 +  population. This highly specific distribution of AC133 makes it exceptionally useful as a reagent for isolating and characterizing human hematopoietic progenitor and stem cells. Cells selected for expression of AC133 antigen can be further purified by selection for other hematopoietic stem cell and progenitor cell markers.

TECHNICAL FIELD

[0001] This invention relates to antigens expressed by hematopoieticstem cells and progenitor cells and to methods of using such antigens,especially for cell separation and purification. Background

[0002] The high turnover of mammalian blood cells requires a supply ofhematopoietic stem cells that are able to give rise to other blood celllineages. The immediate progeny of the hematopoietic stem cell arecalled progenitor cells, and are capable of giving rise to various celltypes within one or more lineages, i.e. the erythroid, myeloid andlymphoid lineages. The stem cell and progenitor cell populationsconstitute only a small percentage of the total number of cells in bonemarrow, fetal liver, etc. These populations are of immense interestbecause of their ability to repopulate the hematopoietic system.

[0003] A number of methods have been described in the literature for thepurification or enrichment of hematopoietic stem cell and progenitorcell populations. There is significant commercial interest in thesemethods because hematopoietic progenitors have a number of clinicaluses. Progenitor cell transplantation is currently used in conjunctionwith chemotherapy and radiation for the treatment of leukemia, breastcancer and other tumors. Frequently, autologous transplants are used toavoid the danger of graft rejection, but there is an increased risk ofdisease reappearance, due to the presence of tumor cells in theengrafting cell population. Transplantation of a more purified source ofprogenitor cells is therefore preferable.

[0004] There is also interest in the use of hematopoietic progenitorcells as a vehicle for gene therapy. Although not yet proven in theclinic, the longevity of hematopoietic stem cells and the disseminationof their progeny in the vasculature are desirable characteristics. Anumber of vectors, including several retrovirus and adenovirus basedconstructs, that can transfect hematopoietic stem cells have beendescribed.

[0005] Proteins and other cell surface markers found on hematopoieticstem cell and progenitor cell populations are of great interest, as theyare useful in preparing reagents for identification, separation andisolation of these populations and in the further characterization ofthese important cells. Although some antigens are now known that can beused in the identification and separation (positive and negative) ofstem cells, such as (for example) the CD 34 antigen, which is found onstem cells but not on mature blood cells, there is a continued need fordevelopment of other antigens, particularly one that can simplify theidentification and separation of desirable classes and subclasses ofcells, especially hematopoietic stem cells and progenitor cells.

BACKGROUND LITERATURE

[0006] U.S. Pat. No. 5,061,620 describes a substantially homogeneoushuman hematopoietic stem cell composition and the manner of obtainingsuch composition. Stromal cell-associated hematopoiesis is described byPaul et al. (1991) Blood 77.:1723-1733. The phenotype of stem cells withrhodamine staining is discussed in Spangrude and Johnson (1990) P.N.A.S.87:7433-7437. Cell surface antigen expression in hematopoiesis isdiscussed in Strauss et al (1983) Blood 61:1222-1231 and Sieff et al.(1982) Blood 60:703-713. Descriptions of pluripotential hematopoieticcells are found in McNiece et al. (1989) Blood 74:609-612 and Moore etal. (1979) Blood Cells 5:297-311. Characterization of a humanhematopoietic progenitor cell capable of forming blast cell-containingcolonies in vitro is found in Gordon et al. (1987) J. Cell. Physiol.130:150-156 and Brandt et al. (1988) J. Clin. Invest. 82:1017-1027. Theuse of progenitor cells in transplantation is discussed in To et al. inProgenitor Threshold in Transplantation (ISBN 1-880854 17-1) pp. 15-20.Utilities for the cell compositions obtained using the methods andcompositions of the invention are described in these publications, amongothers.

[0007] The use of high-gradient magnetic separation for the isolation ofhuman hematopoietic progenitor cells is described in Thomas andLandsdorp (1992) in Advances in Bone Marrow Purging pp.537-54⁴; and Katoand Radbruch (1993) Cytometry 14:384-392. Other methods of magneticselection for human hematopoietic progenitor cells are described inBigas et al. (1992) in Advances in Bone Marrow Purging pp.545-551; Okuet al (1992) in Advances in Bone Marrow Purging pp. 553-560; andHardwick et al. (1992) in Advances in Bone Marrow Purging pp. 583-589.High gradient magnetic cell sorting is described in Miltenyi et al.(1990) Cytometry 11:231-238. Molday, U.S. Pat. No. 4,452,773 describesthe preparation of magnetic iron-dextran microspheres and provides asummary describing the various means of preparation of particlessuitable for attachment to biological materials.

SUMMARY OF THE INVENTION

[0008] Methods and compositions are provided for the enrichment andcharacterization of human hematopoietic progenitor and stem cells. Anantigen has been identified, referred to here as the AC133 antigen, thatis present on stem cells and on progenitor cells and that can be usedfor the identification and/or separation of these important cells fromthe vast majority of cells present in a biological (or other) source ofhematopoietic cells. Novel antigen compositions and reagents that reactwith them, such as antibodies, are provided for use in the methods ofthe invention and for the further investigation of hematopoieticprogenitor and stem cell biology. For example, hematopoietic cells canbe obtained from various sources, including fetal and adult bone marrow,cytokine mobilized peripheral blood cells, and fetal liver, and can beseparated using reagents and methods of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

[0009] The invention now being generally described, the same will bebetter understood by reference to the following description of specificembodiments together with the figures that form part of the currentspecification, wherein:

[0010]FIG. 1 shows a dot-plot from fluorescence activated cell sorting(FACS) analysis of fetal liver cells. The y axis represents cellstaining with AC133 antibody conjugated to phycoerythrin (PE). The cellswere counterstained with HPCA2-FITC (anti-CD34). The numbers representthe percent of total cells that fall within the quadrants.

[0011]FIG. 2 is a graph showing FACS analysis of AC133 antigenexpression on phorbol myristate acetate (PMA) activated Y79.1 cells.

[0012]FIG. 3 is a graph showing FACS analysis of AC133 antigen and CD34expression on PMA activated Y79.1 cells.

[0013]FIGS. 4A and 4B show dot-plots from 3 color FACS analysis of theantibodies AC133, CD38 and HLA-DR on fetal liver cells. The x axis inFIG. 4A represents HLA-DR-FITC, and they axis represents cell stainingwith AC133-PE. The x axis in FIG. 4B represents CD38-FITC, and they axisrepresents cell staining with AC133-PE.

[0014]FIGS. 5A, 5B, 5C and 5D show dot-plots from FACS analysis of theantibodies CD38, HLA-DR, CD90 and CD117 on AC133 positive cells purifiedfrom fetal liver. In FIG. 5A the x axis represents CD38-FITC staining,and the y axis represents HPCA2-PE staining. In FIGS. 5B, 5C and 5D, thex axis represents staining with HPCA2-FITC. The y axis in FIG. 5Brepresents cell staining with anti-HLA-DR-PE. The y axis in FIG. 5Crepresents cell staining with anti-CD90-PE. The y axis in FIG. 5Drepresents cell staining with anti-CD117-PE. The numbers represent thepercent of total cells that fall within the boxed gates.

[0015]FIG. 6 is a gel showing the results of an immunoprecipitation withAC133 antibody and the cell lines KG1a and Y79.1. The lanes are asfollows: 1) molecular weight markers; 2) a 1:5 dilution ofunprecipitated KGla lysate; 3) a 1:50 dilution of unprecipitated Y79.1lysate; 4) KG1a lysate precipitated with AC101 antibody (CD34); 5) Y79.1lysate precipitated with AC101 antibody; 6) KG1a lysate precipitatedwith HPCA2 antibody (CD34); 7) Y79.1 lysate precipitated with HPCA2antibody; 8) KG1a lysate precipitated with 16D11 antibody (CD34); 9)Y79.1 lysate precipitated with 16D11 antibody; 10) KG1a lysateprecipitated with AC133 antibody; 11) Y79.1 lysate precipitated withAC133 antibody; 12) mixed kG1a and Y79.1 lysate precipitated with amixture of AC133 and HPCA2 antibodies; 13) KG1a lysate precipitated with8A3 (anti-CD109) antibody; 14) KG1a lysate precipitated with 15G5(anti-CD109) antibody.

[0016]FIGS. 7A and 7B are graphs showing FACS analysis of CD56 (FIG. 7A)and AC133 antigen (FIG. 7B) expression on PMA activated, tunicamycintreated or untreated Y79.1 cells.

[0017]FIG. 8 is a dot-plot showing FACS analysis of HPCA2-PE (y axis)staining of AC133 magnetically purified fetal liver cells. The x axisrepresents staining with a glycophorin A-FITC conjugate.

[0018]FIGS. 9A and 9B are dot plots showing FACS analysis of HCPA2staining of buffy coat peripheral blood mononuclear cells before andafter AC133 magnetic separation. The y axis shows staining withHCPA2-PE, the x axis shows staining with anti-CD45 and anti-CD15-FITCconjugated antibodies.

[0019]FIG. 10 is a bar graph showing the cloning efficiency of AC133 andAC101 purified cells in a clonogenicity assay.

[0020]FIG. 11 is a bar graph showing the plating efficiency of AC133purified cells and AC133 negative, CD34 positive cells.

[0021]FIG. 12 is a chemical formula showing DNA and amino acid sequencesfor the AC133 antigen.

[0022]FIG. 13 is a schematic diagram of the transmembrane and otherregions of the AC133 antigen.

DESCRIPTION OF SPECIFIC EMBODIMENTS

[0023] Methods and compositions are provided that have use in theenrichment and/or characterization of human hematopoietic stem cellsand/or progenitor cells. The immediate progeny of the hematopoietic stemcell, called here “progenitor” cells, are capable of giving rise tovarious cell types within one or more lineages. In the presentinvention, stem cells and/or a sub-set of progenitor cells (i.e., CFU-GMcells that are needed for short-term ingraftment) can be identified orselected through the use of reagents that specifically bind to a newlydiscovered antigen referred to here as the AC133 antigen (Ag) that ishighly specific for these cells. The high tissue specificity of AC133antigen expression is particularly advantageous during enrichment forhighly purified progenitor cell populations. An AC133-positive cellpopulation is highly enriched for cells that are active in assaysmeasuring progenitor cell activity, particularly in the CFU-GM activity.The subset of cells that is AC133 negative and CD34 positive is enrichedfor BFU-E activity, a measure of erythroid-committed progenitor cellactivity.

[0024] Reagents that specifically bind to the AC133 antigen includewithout limitation physiological ligands, synthetic ligands, polyclonalantibodies, and monoclonal antibodies. An AC133 monoclonal antibody isany monoclonal antibody which interacts specifically with the AC133 cellantigen expressed on a subset of hematopoietic progenitor cells derivedfrom human bone marrow, fetal bone marrow and liver, cord blood andadult peripheral blood. The subset of progenitor cells recognized byantibodies directed to AC133 are CD34^(bright) and containssubstantially all of the CFU-GM activity present in the CD34+subset (aswell as those cells that are still stemcells collected in a collectionof progenitor cells). For purposes of transplantation, cells active inCFU-GM are of particular interest because they provide for production ofneutrophils. Use of an AC133 antibody allows positive immunoselection ofhematopoietic progenitor cell populations, as well as the phenotypicanalysis of progenitor cell populations using flow cytometry. Inparticular, an antibody against AC133 recognizes not just CFU-GM cells,which are needed for short-term engraftment and protection from sepsis,but also primitive long-term re-populating cells that are necessary forlong-term engraftment. Cells selected for expression of AC133 antigencan then be further purified and/or separated by selection for otherhematopoietic stem cell and progenitor cell markers.

[0025] As outlined below in detail, molecules of interest in the variousmethods of the invention include the AC133 antigen itself, reagents thatspecifically bind to AC133 or a fragment thereof, AC133 complexed to aligand, an AC133-ligand complex wherein the ligand is an antibody,nucleic acid sequences encoding the AC133 antigen, and population ofcells that express the AC133 antigen or any of its fragments. The AC133antigen can be isolated from natural sources or produced usingrecombinant DNA technology. The nucleic acids can be cDNA, RNA, agenomic sequence, or a synthetic sequence comprising the coding sequenceby itself or in conjunction with transcriptional regulatory regions andother sequences found in expression and/or cloning vectors. The AC133 Agitself can be obtained in a purified form by isolation from cells, whichcan be identified as positive by AC133 antibody binding using affinitybinding methods known in the art. Positive identification is availableby proteolytic digestion of cell membrane proteins and comparison ofsequences to the protein sequence for AC133 set out in FIG. 12.

[0026] mAb AC133 is an antibody with specificity for a novel cellsurface antigen that is expressed on bright CD34⁺ cells. The antigen isexpressed on a subset of ematopoietic progenitor cells derived fromhuman bone marrow, fetal bone marrow and liver, cord blood, and adultperipheral blood. mAb AC133 can be used in a magnetic bead system toimmunoselect hematopoietic progenitor cell populations, resulting inpotential therapeutic benefit, as well as in the phenotypic analysis ofprogenitor cell populations using flow cytometric techniques. To furthercharacterize the nature of this novel molecule, the AC133 antigen waspurified by immunoaffinity chromatography. The AC133 antigen consists ofa single polypeptide chain with a reduced molecular weight of about 120kD, and comprises a glycoprotein with an about 20-kDaN-glycosidic-linked polysaccharides. The reduced AC133 antigen isrecognized by mAb AC133, suggesting a linear epitope or a sugar epitope.

[0027] It will be recognized by those experienced in the field ofglycoproteins that such molecules are not expected to have exactlyidentical sugar structures because of the enzymatic nature of sugarsynthesis, which occurs without the template (i.e., messenger RNA) thatexists for peptide synthesis, although similarities will certainly existamong the sugar structures in a collection of AC133 molecules because ofsynthesis from the same starting peptide structure. Accordingly, “AC133antigen” refers to proteins having the peptide structure shown in FIG.12 (discussed below) with sugar structures attached at glycoslyationsites. Because of the natural variations in sugar structures, a range ofmolecular weights for glycosylated molecules is also to be expected andcomes within the scope of the present invention. In the case of AC133antigen, there appears to be relatively low variation in the structureand size of the attached sugar residues, compared to other knownproteins. Molecular weight of AC133 is typically found to be in therange of 115 to 127 kD, regardless of the details of the experimentaltechnique used to measure molecular weight.

[0028] The purified AC133 antigen was digested with lysyl endopeptidaseto generate peptides that were isolated by reverse phase HPLC andsequenced by Edman degradation. These peptides were used to designdegenerate oligonucleotides used in the polymerase chain reaction with aWERI-Rb-1 cDNA library template. This technique yielded 1.7 kB ofunambiguous sequence which was then used to isolate the entire cDNAclone. This cDNA encodes a single open reading frame of 2598nucleotides, and predicts a 865 amino acid protein with a molecularweight of 96.8 kDa, which corresponds with the about 90 kDa molecularweight found for the deglycosylated antigen. Hydrophobicity andtransmembrane helicity analysis suggests the presence of fivetransmembrane domains, resulting in two large extracellular loops. Thereare a total of 8 consensus sequences for sites of N-linkedglycosylation, all on the two large (260 and 290 a.a.) loops supportingour proposed structural model with two large extracellular loops and a50 amino acid C-terminal cytoplasmic tail. A truncated version of theAC133 antigen missing the cytoplasmic tail is still recognized by mAbAC133. There are consensus sequences for a leucine zipper motif in bothextracellular loops, which can be involved in receptor interaction withits physiological ligand. As shown in FIG. 13, the AC133 antigen appearsas a 5-transmembrane protein (“5TM protein”) with an extracellularN-terminus and a cytoplasmic C-terminus.

[0029] Families of 4TM (also called tetraspan), 7TM, and 11TM proteinshave been characterized in the literature. While the function of thetetraspan family is not known, the 7TM proteins are generally believedto be G-protein coupled receptors binding chemotactic agonists, and 11TMproteins represent a family of ion-channel receptors. However, a 5TMmolecule has not previously been described, and the structure of theAC133 antigen differs markedly from known 7 TM family members withrespect to molecular weight and size of extracellular loops.Additionally, the AC133 antigen does not share sequence homology with4TM or 7TM proteins, while family members do share significant homologywith each other, particularly within the transmembrane domains.

[0030] Short fragments of the AC133 gene are present in Genbank as EST's(expressed sequence tags), such as adult retina, pancreatic islets andfetal brain. Expression of the AC133 antigen, however, appears to belimited to primitive hematopoietic stem cells and someneural-crest-derived tissues. AC133 antigen is also expressed on NT-2teratocarcinoma cells; however expression is lost as these cellsterminally differentiate into neurons. The interaction of thephysiological ligand with the AC133 antigen (receptor) can provide forintracellular signalling.

[0031] The original monoclonal antibody discovered to the AC133 antigenis one of a panel of antibodies which defines a novel antigen ofapproximately Mr 120,000 which is selectively expressed on CD34^(bright)human hematopoietic stem and progenitor cells. CD34^(bright) cellssupport long-term B cell lymphopoiesis and myelopoiesis in vitro andmediate T, B, myelomonocytic and megakaryocytic repopulation in vivo.CD34^(dim) cells have failed to provide long-term hematopoietic activityin vitro or in vivo. The CD34^(bright) population contains all of theprimitive stem cell activity and therefore is the population of choicefor further studies in hematopoietic stem cell transplantation and genetherapy. AC133 antibody provides a means for the positive selection andphenotypic analysis of hematopoietic stem cells and a subset ofcommitted progenitor cells. The original specific antibody AC133, amurine IgG₁ antibody, was elicited from mice immunized with purifiedCD34⁺ human progenitor cells. In order to determine the precise antigenphenotype of AC133 positive cells, AC133 and CD34 double positive cellswere examined in fetal liver, fetal and adult bone marrow, cord bloodand peripheral blood using 3 and 4 color FACS analysis. The subsetrecognized by AC133 antibody in all tissues are CD₃₄ ^(bright)CD38^(−/+), HLA-DR^(+/−). The CD90⁺, CD117⁺ and CD109⁺ stem cellpopulations are included within the AC133 positive population. TypicallyAC133 stains 20-60% of all CD34⁺ cells, a population which contains allthe non-lineage committed CD34⁺ population as well as CD34⁺ cellscommitted to the granulocyte/monocytic pathway. AC133 antigen expressionhas not been demonstrated on peripheral blood mononuclear cells,granulocytes, platelets or umbilical vein-derived endothelial cells bystandard FACS procedures. FACS analysis on a panel of 25 50 human celllines shows that only 2 retinoblastoma cell lines, Y79.1 and WERI-Rb-1,express AC133 antigen, along with NT-2 teratocarcinoma cells.Transplantation of AC133 positive cells into fetal sheep hasdemonstrated the engrafting capability of selected cells, and humancells which have homed to the fetal sheep bone marrow have beenharvested and shown to engraft secondary recipients, proving the longterm repopulating potential of selected cells. The AC133 gene codes fora polypeptide consisting of 865 aa with a predicted size of 96.8 kDa.This protein has a unique structure, traversing the membrane 5 times.The AC133 antigen therefore defines a new class of mammalian 5TMmembrane proteins. together these data demonstrate that AC133 providesan alternative antigen system for the identification and separation ofhematopoietic stem cells.

[0032] Antibodies that selectively bind to stem cells and/or progenitorcells are of particular interest. Antibodies to AC133 Ag can be obtainedby immunizing a xenogeneic immunocompetent mammalian host (such as amurine, rodentia, lagomorpha, ovine, porcine, or bovine, host) withhuman hematopoietic progenitor cells. The choice of a particular host isprimarily one of convenience. A suitable progenitor cell population forimmunization is obtained by isolating CD34⁺ cells fromcytokine-mobilized peripheral blood, bone marrow, fetal liver, or othersource of progenitor cells. The cells can be incubated withhytohemagglutinin prior to their use as an immunogen.

[0033] Immunizations are performed in accordance with conventionaltechniques, where the cells can be injected subcutaneously,intramuscularly, intraperitoneally, intravascularly into a host.Normally, from about 10⁶ to 10⁸ cells will be used, which can be dividedinto 1 or more injections, usually not more than about 8 injections,over a period of from about one to three weeks. The injections can occurwith or without adjuvant; examples of adjuvant include complete orincomplete Freund's adjuvant, specol, and alum.

[0034] In a preferred embodiment, contralateral immunization is used, asdescribed in the examples below. This method relies on the traffickingability of immune lymphocytes to home to the site of antigenstimulation. The animals are pre-immunized at a localized site on oneside of the body, such as a left footpad, with cells that express manyimmunodominant but irrelevant antigens. Various mature hematopoieticcells can be used for this purpose. The immunogen of interest isinjected at a localized site on the opposite side of the animal.Lymphocytes pre-immunized with and responding to irrelevant antigens aredecoyed to the left-hand draining lymph nodes, while the lymphocytesresponding to the immunogen of interest will be present in theright-hand draining lymph nodes, e.g. the popliteal lymph nodes forfootpad injection. This popliteal lymph node can be used as a source ofcells for fusion.

[0035] After completion of the immunization schedule, the antiserum canbe harvested in accordance with conventional techniques to providepolyclonal antisera specific for the surface membrane proteins ofhematopoietic progenitor cells, including AC133 Ag. Lymphocytes can thenbe harvested from the appropriate lymphoid tissue, e.g. spleen ordraining lymph node, and fused with an appropriate fusion partner,usually a myeloma line, to produce a hybridoma secreting a specificmonoclonal antibody. Screening clones of hybridomas for the antigenicspecificity of interest can be performed in accordance with conventionalmethods.

[0036] Of particular interest are the specific monoclonal antibody AC133described in the Examples below; other antibodies (both monoclonal andpolyclonal) that bind to the AC133 antigen, especially cross-reactiveantibodies (i.e., those which bind to the same epitope, andsubstantially inhibit simultaneous binding); species analogs thereof;binding fragments thereof; and conjugates thereof. A deposit of a murinehybridoma cell line that expresses an antibody to the AC133 antigen wasmade at the American Type Culture Collection, 10801 UniversityBoulevard, Manassas Va. 20110, on Apr. 23, 1997, and given the ATCCdesignation HB12346. These antibodies are capable of immunoselection forthe hematopoietic subset of interest.

[0037] It is known that antibodies can be produced as a single chaininstead of a normal multimeric structure. Single chain antibodies aredescribed in Jost et al. (1994) J.B.C. 269:26267-73, and in numerousother publications. DNA sequences encoding the variable region of theheavy chain and the variable region of the light chain are ligated to aspacer encoding at least about 4 amino acids of small neutral aminoacids, including glycine and/or serine. The protein encoded by thisfusion allows assembly of a functional variable region that retains thespecificity and affinity of the original antibody.

[0038] Methods of humanizing antibodies are also known in the art. Ahumanized antibody can be the product of an animal having transgenic,human, immunoglobulin-constant-region genes (see for exampleInternational Patent Applications WO 90/10077 and WO 90/04036).Alternatively, the antibody of interest can be engineered by recombinantDNA techniques to substitute the CHI, CH2, CH3, hinge domains, and/orthe framework residues with the corresponding human sequence (see WO92/02190).

[0039] The use of Ig cDNA for construction of chimeric immunoglobulingenes is known in the art (Liu et al. (1987) P.N.A.S. 84:3439 and (1987)J. Immunol. 159:3521). In these techniques mRNA is isolated from ahybridoma or other cell producing the antibody and used to produce cDNA.The cDNA of interest can be amplified by the polymerase chain reactionusing specific primers (U.S. Pat. Nos. 4,683,195 and 4,683,202).Alternatively, a library can be made and screened to isolate thesequence of interest. The DNA sequence encoding the variable region ofthe antibody is then fused to human constant region sequences. Thesequences of human constant region genes can be found in Kabat et al.(1991) Sequences of Proteins of Immunological Interest, N.I.H.publication no. 91-3242. Human C region genes are readily available fromknown clones. The chimeric, humanized antibody can then be expressed byconventional methods.

[0040] Antibody fragments, such as Fv, F(ab′)₂ and Fab fragments, can beprepared by cleavage of the intact antibody, e.g. by protease orchemical cleavage. Alternatively, a truncated gene can be designed. Forexample, a chimeric gene encoding a portion of the F(ab′)₂ fragmentcould include DNA sequences encoding the CHI domain and hinge region ofthe H chain, followed by a translational stop codon to yield a truncatedantibody fragment.

[0041] Antibodies to the AC133 antigen bind to a protein that has anapparent molecular weight (under Western blot conditions from reducingSDS-PAGE gels, based on commercially available standards) of about 120kD, and generally appears to be in the range of about 115 to 127 kD. Theantibody appears to recognize a sugar epitope, as AC133 antibody cannotbe immunoprecipitated from tunicamycin-treated WERI-Rb-1 cells. TheAC133 antigen is expressed on a subset of CD34⁺ cells, but is absent onendothelium and fibroblasts. Included in the population ofAC133-positive cells are HLA-DR+, CD90⁺ and CD117⁺ progenitor cells (theantigen formerly known as CD90⁺ is now known as CD90⁺; both DR positiveand negative as well as CD38 positive and negative cells are included inthis population). This population contains substantially all of thehematopoietic stem activity present in the CD34⁺ subset of hematopoieticcells.

[0042] Reagents that specifically bind to the AC133 antigen are notlimited to antibodies. Any of numerous methods known in the art todetect the binding of one species to another can be used to assay forthe presence of an AC133 antigen-binding reagent. One universallyadaptable assay involves distribution of radioactivity between solubleand solid phases can be detected using radioactively labeled testcompounds and AC133 antigen attached to a solid phase. AC133 antigen canbe attached, for example, to a solid phase in a column, and a tritium-or ¹⁴C-labelled test compound in a physiological buffer can be passedthrough the column. Bound radioactivity can be detected directly on thecolumn or by subtraction of radioactivity in the soluble phase passingthrough the column from the applied radioactivity. Binding affinity canbe detected by measuring levels of bound radioactivity at differentconcentrations of test compound after allowing sufficient time forbinding to equilibrate. Specificity of binding for AC133 can be detectedby determining whether test compounds that bind to AC133 also bind toantigens present on mature blood cells (or other antigens of interest ina preselected assay medium). Especially preferred ligands are those thatare selective for AC133 with less than 10%, preferably less than 5%,crossreactivity with any antigen present on mature blood cells.Crossreactivity can be measured by any standard technique and preferablyis measured by a competitive binding assay between pure AC133 antigen,the ligand to be tested, and the suspected crossreactive antigen using aconcentration of AC133 antigen and test ligand where the ligandhalf-saturates binding to AC133. Most preferably, crossreactivity ismeasured at a concentration of AC133 antigen that half saturatesmonoclonal antibody ATCC HB12346 when the antibody is present at aconcentration of 50 ng/100 μI.

[0043] Once a reagent is identified that specifically binds to AC133,the reagent (in its radioactively labeled form, in a non-radioactiveform modified to contain another label, or in certain uses in unlabeledform) can be used in various assays or biological uses that call for thebinding of a reagent to AC133, such as fluorescent staining, cellseparation, or cell differentiation, either in vivo and in vitro. Forexample, immunoselection with an antibody against AC133 provides a meansof purifying hematopoietic progenitor and stem cells. The antibodiesalso find use in diagnostics to detect or enumerate hematopoieticprogenitor cells, in dividing the CD34 positive population intofunctionally distinct sub-populations, in isolation of progenitor cells,and in preparation of progenitors to produce mature blood cells.Biological samples (e.g. blood or derivatives thereof, biopsies, andsynovial fluid) can be assayed for the presence of cells expressing thesurface molecule bound by the subject antibodies. For example, assayscan be performed on cell lysates, intact cells, or frozen sections inorder to distinguish different types of cells.

[0044] The subject antibodies and other reagents that specifically bindto AC133 are useful for the preparation of substantially pure humanhematopoietic progenitor and stem cells. A subset of progenitor cellscan be separated from other hematopoietic cells on the basis of AC133binding and can be further separated from each other by binding to othersurface markers known in the art. Sources of hematopoietic cells includefetal or adult bone marrow; fetal liver; umbilical cord blood; andperipheral blood, particularly cytokine mobilized peripheral blood (see,for example, Campos et al. (1993) Leukemia 7:1409-15 and Grigg et al(1993) Bone Marrow Transplant 11, Suppl 2:23-9).

[0045] Human stem cells have been reported to have the phenotypeCD34^(bright); HLA-DR⁺; CD38^(dim/negative); CD117(c-kit)^(dim);CD90(Thy-1)⁺; and to lack expression of a variety of lineage specificmarkers, including CD3, CD4, CD7, CD8, CD14, CD15, and CD19. A negativedesignation indicates that the level of staining is at or below thebrightness of an isotype-matched negative control. A dim designationindicates that the level of staining may be near the level of a negativestain, but may also be brighter than an isotype matched control.

[0046] Procedures for separation include magnetic separation usingantibody-coated magnetic beads and affinity chromatography or “panning”using antibody attached to a solid matrix (e.g. plate). Techniquesproviding accurate separation include fluorescence-activated cellsorters, which can have varying degrees of sophistication, such ashaving multiple color channels, low angle and obtuse light scatteringdetecting channels, or impedance channels. Dead cells can be eliminatedby selection with dyes associated with dead cells e.g., (propidiumiodide, LDS). Red blood cells can be removed by (for example)elutriation, hemolysis, or Ficoll-Paque gradients. Any technique can beemployed that is not unduly detrimental to the viability of the selectedcells.

[0047] Conveniently, antibodies can be conjugated with labels for anumber of different purposes: e.g., magnetic beads to allow for ease ofseparation of a particular cell type; biotin, which binds with highaffinity to avidin or streptavidin; fluorochromes, which can be usedwith a fluorescence activated cell sorter; haptens; and the like.Multi-color analyses can be employed with a FACS or in a combination ofimmunomagnetic separation and flow cytometry. Multi-color analysis is ofinterest for the separation of cells based on multiple surface antigens:e.g., AC133⁺, CD90⁺ or CD117⁺, AC133⁻, or CD34⁺. Fluorochromes whichfind use in a multi-color analysis include phycobiliproteins, e.g.phycoerythrin and allophycocyanins; fluorescein, and Texas red.

[0048] In one embodiment of the invention, an anti-AC133 antibody isdirectly or indirectly conjugated to a magnetic reagent, such as asuperparamagnetic microparticle (microparticle). Direct conjugation to amagnetic particle is achieved by use of various chemical linking groupsas known in the art. For example, antibody can be coupled to themicroparticles through side chain amino or sulfhydryl groups andheterofunctional cross-linking reagents. A large number ofheterofunctional compounds are available for linking to entities. Apreferred linking group is 3-(2-pyridyldithio)propionic acidN-hydroxysuccinimide ester (SPDP) or4-(N-maleimidomethyl)-cyclohexane-1-carboxylic acid N-hydroxysuccinimideester (SMCC) with a reactive sulfhydryl group on the antibody and areactive amino group on the magnetic particle.

[0049] Alternatively, an anti-AC133 antibody is indirectly coupled tomagnetic particles. The antibody is directly conjugated to a hapten, andhapten-specific, second-stage antibodies are conjugated to theparticles. Suitable haptens include digoxin, digoxigenin, FITC,dinitrophenyl, nitrophenyl, avidin, and biotin. Methods for conjugationof the hapten to a protein are known in the art, and kits for suchconjugations are commercially available.

[0050] For separation or identification of stem cells or progenitorcells, an antibody is added to a hematopoietic cell sample. The amountof an anti-AC133 antibody necessary to bind a particular cell subset isempirically determined by performing a test separation and analysis. Thecells and an anti-AC133 Ab are incubated for a period of time sufficientfor complexes to form, usually at least about five minutes, more usuallyat least about 10 minutes, and usually not more than one hour, moreusually not more than about 30 minutes.

[0051] The cells can additionally be incubated with antibodies orbinding molecules specific for cell-surface markers known to be presentor absent on hematopoietic progenitor or stem cells. For example, CD90,CD117 and HLA-DR are useful in the positive selection of stem cells.Various markers known to be absent on stem cells, such as CD3, CD4, CD8,CD14, CD15, and CD19, can be used for negative selection. The labeledcells are separated in accordance with the specific antibodypreparation. Fluorochrome-labeled antibodies are useful for FACSseparation and magnetic particles for immunomagnetic selection orparticularly high gradient magnetic selection (HGMS). Exemplary magneticseparation devices are described in WO/90/07380, PCT/US96/00953 and EP438,520, herein incorporated by reference.

[0052] The purified cell population can be collected in any appropriatemedium. Various media are commercially available and can be used,including Dulbecco's Modified Eagle Medium (DMEM), Hank's Basic SaltSolution (HBSS), Dulbecco's phosphate buffered saline (DPBS), RPMI,Iscove's modified Dulbecco's medium (IMDM), and phosphate bufferedsaline (PBS) with 5 mM EDTA, any of which can be supplemented with fetalcalf serum (FCS), bovine serum albumin (BSA), or human serum albumin(HSA).

[0053] Compositions highly enriched for human hematopoietic progenitorand/or stem cells (depending on the source of cells) are achieved inthis manner in a single step. The desired cells will be at or about 80%or more of the cell composition, and preferably be at or about 90% ormore of the cell composition. Specific populations of interest includeAC133⁺ cells, which are characterized as CD34^(bright) and HLA-DR^(+/−).This population can be further selected for those cells that are CD90⁺,CD117⁺ and/or CD38^(dim). Functionally these cells are highly enrichedfor CFU-GM activity and for long-term re-populating cells. Anotherpopulation of interest is CD133⁻ and CD34⁺, which is enriched for BFU-Eactivity. The use of the subject antibodies for purification areadvantageous over the use of CD34, because AC133 is expressed by a morerestricted population of cells, thereby permitting a more enrichedsubset for the specific activity of interest.

[0054] Once the desired cells have been isolated, they can be propagatedby growing in conditioned medium from stromal cells, co-culturing withsuch stromal cells, or in media comprising maintenance factorssupporting the proliferation of such progenitor cells e.g., stem cellfactor or combinations of interleukins. The medium employed forculturing cells is conveniently a defined enriched medium, such as IMDMor a mixture of IMDM and RPMI 1640, and will generally be composed ofsalts, amino acids, vitamins, 5×10⁻⁵ M θ-mercaptoethanol,streptomycin/penicillin and 10% fetal calf serum, and can be changedfrom time to time, generally at least once to twice per week.

[0055] The subject cell compositions find use in a variety of ways. Theycan be used to reconstitute an irradiated host and/or a host subject tochemotherapy. By providing for maturation, proliferation anddifferentiation into one or more selected lineages through specificdifferent growth factors the progenitor cells can be used as a source ofcommitted cells. Such factors as erythropoietin, colony stimulatingfactors (e.g., GM-CSF, G-CSF or M-CSF), interleukins (e.g. IL-1, -2, -3,-4, -5, -6, -7, -8, -9, or -10), or the like, or stromal cells can beused to influence the growth and differentiation of progenitor cells.

[0056] The cells can also be used in the isolation and evaluation offactors associated with the differentiation and maturation ofhematopoietic cells, including reagents that specifically bind to theAC133 antigen. Thus, the cells can be used in assays to determine theactivity of media, such as conditioned media; to evaluate fluids forgrowth factor activity or involvement with dedication of lineages; orthe like.

[0057] The cells can be used for the treatment of genetic diseases.Genetic diseases associated with hematopoietic cells can be treated bygenetic modification of autologous or allogeneic stem cells to correct agenetic defect or treat to protect against disease, e.g., HIV. Forexample, diseases such as θ-thalassemia, sickle cell anemia, adenosinedeaminase deficiency, recombinase deficiency, or recombinase regulatorygene deficiency can be corrected by introduction of the wild-type geneinto the subject cells, either by homologous or random recombination.Alternatively, normal allogeneic progenitor cells can be transplanted.Diseases other than those associated with hematopoietic cells can alsobe treated, where the disease is related to the lack of a particularsecreted product such as hormone, enzyme, interferon, factor, or thelike.

[0058] The cells can be frozen at liquid nitrogen temperatures andstored for long periods of time, as they can be thawed and reused. Thecells will usually be stored in 5% DMSO and 95% fetal calf serum. Oncethawed, the cells can be expanded by use of growth factors or stromalcells associated with stem cell proliferation and differentiation.

[0059] The AC133 antigen can be obtained in substantially pure form fromeither natural sources or by recombinant techniques. From naturalsources, the antigen-positive cells are lysed and passed through anaffinity column of anti-AC133 monoclonal antibody. Hematopoieticprogenitor cells can be isolated from natural sources by conventionalseparation techniques, or cell lines described in the experimentalsection can be used as a source of antigen. The affinity-purifiedprotein is eluted from the affinity column with an appropriate saltsolution or aqueous/organic gradient, such as acetonitrile or ethanol,usually in the presence of a low acid concentration, e.g., 0.1-1 percenttrifluoroacetic acid. The eluted protein is then further purified bychromatography, electrophoresis, or the like in accordance withconventional techniques.

[0060] The examples below describe the use of a monoclonal antibody topurify the AC133 antigen by affinity chromatography resulting in greaterthan 95% pure AC133 antigen. Peptides of such a purified preparation canbe prepared and isolated for sequence analysis, as a result of whichnucleic acid probes can be designed for the isolation of AC133 genesequences. The gene sequence of AC133 set forth herein (FIG. 12) allowsthe antigen to be obtained by recombinant techniques. For example, totalRNA is isolated from cells that have been shown by antibody binding toexpress the targeted protein. Residual DNA is removed in accordance withconventional techniques, and the polyadenylated RNA can be purifiedfurther, for example on oligo-dT sepharose or by gel chromatography.cDNA is then prepared in accordance with conventional techniques usingreverse transcriptase (see Sambrook et al., supra and the Examplesbelow). The cDNA is then introduced into an appropriate cloning system,such as Σ gt11, where the cDNA is expressed. The phage plaques can thenbe screened using the subject antibodies, or by employing polyclonalantisera. Alternatively, a cloning system can be used which allowsprobing with nucleic acid sequences derived from the AC133 antigenprotein sequence. The cDNA inserts are then subcloned into othervectors, as desired. The cDNA can be used for further probing of thecDNA library for a complete transcript. Alternatively, the cDNA sequencecan be used to probe a genomic library to identify the genomic geneencoding the subject proteins (See, for example, Molecular Cloning: ALaboratory Manual, 2nd ed., J. Sambrook, E. F. Fritsch, T. Maniatis,CSHL, Cold Spring Harbor, N.Y., 1989).

[0061] DNA of the invention includes the nucleotide sequences encodingthe AC133 protein or fragments thereof, as well as adjacent 5′ and 3′non-coding nucleotide sequences involved in the regulation of expressionof the protein encoded by the genes, and will include up to about thelength of the mature mRNA or genomic DNA. Thus, the present inventionprovides an isolated nucleic acid molecule, in which the moleculecomprises (1) a first sequence having an amino acid coding region forAC133 as set forth in FIG. 1 (SEQ ID NO:1); (2) a second sequence,wherein said second sequence is a subsequence of said first sequence andis at least 14, preferably at least 17 or 20, more preferably at least25, nucleotides in length; (3) a third sequence in which at least onenucleotide of said first or second sequences is replaced by a differentnucleotide; or (4) a fourth sequence complementary to any of said first,second or third sequences; with the proviso that (i) if said molecule isan RNA molecule, U replaces T in said sequence of said molecule, (ii)said third sequence is at least 90%, preferably at least 95%, identicalto said first or second sequence, and (iii) said second sequence is notnucleotides 347-667, 1564-1696, or 2110-2386 of SEQ. ID NO:1. Alsoincluded as DNA of the invention is the corresponding genomic sequence,including introns. These non-coding sequences include terminator andpolyadenylation sequences, regulatory protein binding sequences,transcriptional sequences, and the like. Molecules containing the fulllength AC133 cDNA sequences are useful as sources of subsequences or asstarting materials for the preparation of the AC133 molecule itself.

[0062] A “subsequence” is a group of consecutive nucleotides from thecDNA sequence. Any of these sequences can be used in the identificationof the presence (or absence) of the AC133 gene or of the expression ofmRNA encoding the AC133 antigen. Such subsequences can be prepared bychemical synthesis from starting nucleotides (as in an automated genesynthesizer) or by biochemical manipulation of the full-length sequences(e.g., using restriction endonucleases to prepare fragments, optionallyfollowed by (1) cleavage of terminal nucleotides and exonucleases and/or(2) size sorting and/or affinity capture to select the desiredsequence). Any subsequence of the AC133 sequence described in SEQ IDNo.: 1 of sufficient length to be unique among the other nucleic acidspresent under the conditions being used is useful as one of the twoprimers used in a polymerase chain reaction (PCR) amplification of allor part of the genomic AC133 gene. The length of a subsequence necessaryto hybridize uniquely with the desired target sequence will vary withthe particular method being used, and selection of the length is withinthe ordinary skill of those who carry out routine identification ofgenetic material. A preferred subsequence is at least 15 nt in length,more preferably at least 18 nt, even more preferably at least 19, 20,21, 25, or 30 nt in length up to the full length of the nucleotidesequence shown as SEQ. ID NO:1, preferably less than 200 nt in length ifused as a hybridization probe or less than 50 nt in length if used as aPCR primer.

[0063] Three subsequences within the coding region of SEQ. ID NO: 1 werepreviously recorded in Genbank as EST's of unknown function.Accordingly, these Genbank subsequences, nucleotides 347-667, 1564-1696,and 2010-2386, are not claimed as subsequences of the invention.Additionally, there are a number of EST's in Genbank from the 3′untranslated region of SEQ. ID NO:1, also of unknown function,specifically in the regions covered by nucleotides 2684-3332 and3408-3804. Subsequences from these two regions are not claimed as partof the present invention. Longer subsequences of the entire sequenceshown as SEQ. ID NO:1 that contain one or more of the Genbank sequences,as well as subsequences of any length that include part of one or moreGenbank sequence but also contain newly identified nucleotides set forthin SEQ. ID NO:1, are considered to be part of the present invention.

[0064] The nucleic acid compositions of the subject invention can begenomic or cDNA sequences encoding all or a part of the subject protein.Fragments can be obtained of the cDNA or genomic sequence by chemicallysynthesizing oligonucleotides in accordance with conventional methods,such as by restriction enzyme digestion or by PCR amplification. For themost part, fragments will be of at least 12 nt, more usually at least 18nt, or one of the other lengths described above. Preferred fragmentswill include a functional epitope. The sequence providing for afunctional epitope can be determined by expression of the sequence, andassaying for reactivity of the expression product with specificantibodies by conventional immunoassay.

[0065] Exemplary amino acid and DNA sequences of the invention are setforth in SEQ ID No.: 1 and 2 below. Standard abbreviations fornucleotides and amino acids are used in this specification. Polypeptidesderived from the natural AC133 antigen are particularly preferredembodiments of the invention, although variations based on the specificsequences of these polypeptides are also parts of the present invention.In its broader aspects the invention (as it pertains to polypeptides perse) includes any polypeptide selected from the group consisting of (1) afirst amino acid sequence of AC133 as set forth in SEQ ID NO: 2; (2) asecond amino acid sequence wherein the second sequence is a subsequenceof the first sequences and is at least 6, preferably 8, more preferably10, amino acids in length; or (3) a third sequence in which at least oneamino acid of the first or second sequences is replaced by a differentamino acid, with the proviso that the amino acid replacement is areplacement of one acidic residue for another, one basic residue foranother, one non-polar residue for another, one uncharged polar residuefor another, or one aromatic residue for another, with the proviso thatthe third sequence is at least 90%, preferably 95%, identical to thefirst or second sequence.

[0066] Two amino acid sequences are homologous if there is a partial orcomplete identity between their sequences. For example, 85% homologymeans that 85% of the amino acids are identical when the two sequencesare aligned for maximum matching. Gaps (in either of the two sequencesbeing matched) are allowed in maximizing matching. Gaps of 5 or less arepreferred with 2 or less being more preferred. Alternatively andpreferably, two protein sequences (or polypeptide sequences derived fromthem of at least 30 amino acids in length) are homologous as this termis used herein, if they have an alignment score of more than 5 (instandard deviation units) using the program align with the mutation datamatrix and a gap penalty of 6 or greater (Dayhoff, M. O., in Atlas ofProtein Sequence and Structure, 1972, vol. 5, National BiomedicalResearch Foundation, pp. 101-110, and supplement 2 to this volume, pp.1-10). The two sequences or parts thereof are more preferably homologousif their amino acids are greater than or equal to 50% identical whenoptimally aligned using the align program.

[0067] Minor amino acid variations from the natural amino acid sequencesets forth in SEQ ID No.: 2 are contemplated; in particular,conservative amino acid replacements are contemplated. Conservativereplacements of those that take place within a family of amino acidsthat are related in their side chains. Genetically encoded amino acidsare generally divided into four families: (1) acidic, aspartate,glutamate; (2) basic: lysine, arginine, histidine; (3) non-polar:alanine, valine, leucine, isoleucine, proline, phenylalanine,methionine, tryptophan; and (4) uncharged polar: glycine, asparagine,glutamine, cysteine, serine, threonine, tyrosine. Phenylalanine,tryptophan and tyrosine are sometimes classified jointly as aromaticamino acids. For example, it is reasonable to expect that an isolatedreplacement of a leucine with an isoleucine or valine, an aspartate witha glutamate, a threonine with a serine, or a similar replacement of anamino acid with a structurally related amino acid will not have a majoreffect on the binding properties of the resulting molecule, especiallyif the replacement does not involve an amino acid as a binding siteinvolved in the interaction of AC133 or its derivatives with a reagentthat binds specifically to AC133. Whether an amino acid change resultsin a functional peptide can readily be determined by assaying thespecific binding properties of the polypeptide derivative.

[0068] As shown in FIG. 13, there are a number of regions havingdifferent functions in the peptide structure of AC133. These regions canbe described (beginning with the amino terminus) as an extracellularN-terminus, a first transmembrane region, a first cytoplasmic loop, asecond transmembrane region, a first extracellular loop, a thirdtransmembrane region, a second cytoplasmic loop, a fourth transmembraneregion, a second extracellular loop, a fifth transmembrane, and acytoplasmic C-terminus. Approximate sizes of the regions are shown inFIG. 13, with best estimates of the amino acids present in the differentregions being as follows: extracellular N-terminus, aa 20-107; firsttransmembrane region, aa 107-126; first cytoplasmic loop, aa 127-157;second transmembrane region, aa 158-179; first extracellular loop, aa180-435; third transmembrane region, aa 436-454; second cytoplasmicloop, aa 455-480; fourth transmembrane region, aa 481-503; secondextracellular loop, aa 504-792; fifth transmembrane, aa 793-816; andcytoplasmic C-terminus, aa 817-865. There appears to be a cleavablesignal sequence (aa 1-19) at the amino terminus of the encoded peptide;this sequence is not included as part of the regions shown in FIG. 13but will be present in synthetically produced AC133 peptides.

[0069] Also shown in FIG. 13 are the approximate locations of shortpeptide segments (P1-P4) that were identified to verify the structure ofthe AC133 antigen and of glycosylation sites (indicated by a “Y” at thepoint of attachment). FIG. 12 also shows the glycosylation sites (whichare boxed in the amino acid sequence) and transmembrane regions (whichare underlined). Two glycosylation sites overlap (NNTS, which consistsof an overlapping NNT and NTS) and are shown by a larger box with dashedlines indicating the individual consensus glycosylation sites.

[0070] The DNA sequences can be obtained in substantial purity and canbe obtained as an isolated molecule other than a sequence of an intactchromosome. Usually, the DNA will be obtained substantially free ofother nucleic acid compounds, generally being at least about 50%,usually at least about 90% pure and are typically “recombinant”, i.e.,flanked by one or more nucleotides with which they are not normallyassociated with on a natural chromosome.

[0071] The DNA sequences are used in a variety of ways. They can be usedas probes for identifying related surface proteins in the same or otherspecies. The DNA can also be used to identify cells or organs that areexpressing the subject genes. Techniques in which one probes cells forthe presence of particular nucleotide sequences, particularly as DNA,mRNA or cDNA, are well-established in the literature and do not requireelaboration here. Conveniently, MRNA can be isolated free of DNA, and byusing reverse transcriptase and PCR with specific primers, the subjectcDNAs of interest of can be expanded, separated on gel electrophoresisand then probed using Southern blotting or sequencing. Other techniquescan also find use.

[0072] Homologous sequences are those with substantial sequencesimilarity to AC133 antigen sequences included within the subjectinvention, i.e., at least 80%, preferably at least 90%, more preferablyat least 95%, sequence identity with the nucleotide sequence of thesubject DNA sequence. Sequence similarity is calculated based on areference sequence, which can be a subset of a larger sequence, such asa conserved motif, coding region, or flanking region. A referencesequence will usually be at least about 18 nt long, more usually atleast about 30 nt long, and can extend to the complete sequence that isbeing compared. Such homologous nucleic acid sequences will be detectedby hybridization under low stringency conditions, for example, at 50° C.and 10×SSC (0.9 M NaCl/0.09 M sodium citrate) and remain bound whensubject to washing at 55° C. with 1×XSSC.

[0073] For expression, the DNA sequences can be inserted into anappropriate expression vector, where the native transcription initiationregion can be employed or an exogenous transcriptional initiationregion. The promoter can be introduced by recombinant methods in vitro,or as the result of homologous integration of the sequence into achromosome. A wide variety of transcriptional initiation regions areknown for a wide variety of expression hosts, where the expression hostscan involve prokaryotes or eukaryotes, particularly E. coli, B.subtilis, mammalian cells, such as CHO cells, COS cells, monkey kidneycells, lymphoid cells, particularly human cell lines, and the like.Generally a selectable marker operative in the expression host will bepresent. The promoter can be operably linked to the coding sequence ofthe genes of interest so as to produce a translatable mRNA transcript.Expression vectors have convenient restriction sites located near thepromoter sequence so as to provide for the insertion of nucleic acidsequences encoding heterologous proteins. The promoters in suitableexpression vectors can be either constitutive or inducible. Expressionvectors for the production of fusion proteins, where the exogenousfusion peptide provides additional functionality, such as increasedprotein synthesis, stability, reactivity with defined antisera, or anenzyme marker, e.g., θ-galactosidase, are of particular interest.

[0074] Expression cassettes can be prepared comprising the transcriptioninitiation region, which can be constitutive or inducible, with orwithout an enhancer sequence, including the endogenous or heterologousenhancer sequence, the AC133 gene or fragment thereof, and atranscriptional termination region, optionally having a signal forattachment of a poly A sequence. The gene can be genomic, including thenative introns, or cDNA gene, or portion thereof. Of particular interestis the use of sequences which allow for the expression of functionalepitopes, usually at least about 24 nucleotides in length, more usuallyat least about 48 nucleotides in length, and up to the complete openreading frame of the gene.

[0075] After introduction of the DNA, the cells containing the constructcan be selected by means of a selectable marker, the cells expanded andthen used for expression. Where secretion is desired, a signal peptidecan be joined to the sequence encoding the subject proteins or fragmentsthereof, whereby the protein will be expressed, translocated through thecell membrane, and processed to remove the signal peptide.

[0076] The expression cassettes can be introduced into a variety ofvectors, where the vectors will normally be characterized by the abilityto provide selection of cells comprising the expression vectors. Thevectors can provide for extrachromosomal maintenance, particularly asplasmids in bacteria or viruses in eukaryotic cells, or for integration,particularly in mammalian cells. Where extrachromosomal maintenance isdesired, an origin sequence will be provided for the replication of theplasmid, which can be a low- or high-copy plasmid. A wide variety ofmarkers are available for selection, particularly those which protectagainst toxins, more particularly against antibiotics. The particularmarker which is chosen will be selected in accordance with the nature ofthe host, where in some cases, complementation can be employed withauxotrophic hosts, e.g., yeast. Introduction of the DNA construct can beby any convenient means, e.g., calcium-precipitated DNA,electroporation, fusion, transfection, or infection with viral vectors.

[0077] The following examples are offered by way of illustration and notby way of limitation.

EXAMPLES

[0078] Generation of the AC133 Monoclonal Antibody by ContralateralImmunization.

[0079] Five New Zealand Black (NZB) mice were inoculated a total ofseven times over a twenty day period, via the footpad route, withpurified CD34 positive human progenitor cells, which had beenpre-incubated with phytohemagglutinin (PHA) (Gibco/BRL).

[0080] Mice were pre-immunized on Day -3 in the left hand footpad withcells that express many immunodominant but irrelevant antigens. In thiscase peripheral blood mononuclear cells (PBMC) were used as anirrelevant cell, as they express many antigens such as Class I HLAantigens, HLA-DR, CD15, CD26, CD29, CD31, CD36, CD44, CD45, CD58, etc.,which are also expressed on hematopoietic stem cells. On day 0 PBMC arereinjected into the left footpad, and purified stem cells are injectedinto the right hand footpad. PBMC and purified stem cells arepre-incubated with PHA for ten minutes and washed with PBS prior toinjection. Progenitor cells were isolated from a leukaphoresis pack of acytokine mobilized donor using immunomagnetic beads. This treatmentprovides non-specific adjuvant effects, and obviates the need adjuvantssuch as Freund's. Mice are given a total of 5-8 such injections at threedays intervals.

[0081] On day 21, one day after the last injection, the mouse right handpopliteal lymph nodes were removed. A lymphocyte suspension wasprepared, and the cells fused to SP2/0 Ag14 myeloma cells using amodification of the method originally described by Kohler and Milstein(1975) Nature 256:495-497. Cells were plated on 96 well plates inDMEM+20% fetal calf serum, with 10⁻⁴ M hypoxanthine and 2 μg/mlazaserine (Buck et al. (1984) in Monoclonal Antibodies and FunctionalCell Lines Kennet et al. eds., Plenum Press, New York pp.275-309). Onday 10, visible hybridoma colonies were apparent. Supernatants (s/n)from hybridoma containing wells were screened for binding to a fetalliver cell preparation containing up to 15% CD34+ cells, using a 2 colorflow cytometry assay. Binding of mouse Ig containing s/n to the testcells was traced with rat anti-mouse Ig-conjugated to phycoerythrin(IgPE) and counterstained with a known mouse anti-CD34 antibody (AC101)conjugate. FIG. 1 shows the results from this two color FACS analysisusing AC133 supernatant. AC133 is shown to stain only the bright CD34positive cells in the fetal liver preparation. AC133 hybridoma cellswere shown to secrete an IgG1/kappa antibody. The cells were expanded inculture and stocks frozen in liquid nitrogen. AC133 cells were subclonedby limiting dilution analysis and a series of positively secretingsubclones were also frozen in liquid nitrogen.

[0082] Antibody Purification and Conjugation.

[0083] AC133 cells were initially grown as an ascites tumor in nudemice, with collection of antibody-rich ascites fluid. More recentlyAC133 cells have been grown to very high density in a hollow fiberculture device (Cellmax QUAD artificial capillary system, Cellco Inc.,Germantown, Md.). Pure IgG antibody was 20 prepared from hollow fibercultures or from ascites fluid by Protein A chromatography. Pureantibody was stored in 0.01M phosphate buffered saline (PBS) with 0.01%sodium azide at 4° C. This pure antibody stock was used to preparefluorescein isothiocyanate (FITC) (Wofsy et al. (1980)in SelectedMethods in Cellular Immunology, Mishell and Shiigi eds., W.H. Freemanand Co., San Francisco. pp.294-295), phycoerythrin (PE) (Hardy (1986) inHandbook of Experimental Immunology, Weir et al., eds. BlackwellScientific Press, Ox ford. p.31), or magnetic bead conjugates, accordingto standard protocols.

[0084] AC133 Expression on Normal Tissues and Cell Lines.

[0085] Using standard FACS staining procedures, there was no detectablestaining of peripheral blood mononuclear cells, granulocytes orplatelets, or human umbilical vein endothelial cells with AC133antibody. Examination of a panel of human cell lines by FACS analysis(data shown in Table 1) showed that only three cell lines tested, theretinoblastoma cell lines Y79.1 and WERI-Rb-1 and the teratocarcinomacell line NT-2, expresses detectable levels of AC133 antigen. TABLE 1AC133 Expression on Human Cell Lines Cell Line Cell Type AC133 8402 Tcell line (CD34+) − 8866 B-LCL − AZ676 breast carcinosarcoma − BJAB N.American Burkitts' lymphoma − BT474 breast tumor − BT549 breast tumor −BT20 breast tumor − CaCL74-36 melanoma − Daudi B-LCL- − Du4475 breasttumor (CD34+) − HEL92.1.7 erythroleukemia − HL-60 promyelocytic leukemia− HPB-ALL acute lymphocytic leukemia − HS-R myeloma (EBV+) − HT1080fibrosarcoma − HT29 colon adenocarcinoma − IM-9 B-LCL − JM T cell line −Jurkat T cell line − KG1a acute myclogenous leukemia (CD34+) − KG1 acutemyelogenous leukemia (CD34+) − KG1a.5 acute myelogenous leukemia (CD34+)− K562 erythroleukemia − MOLT-4 T cell line − MCF-7 breast tumor − RajiB-LCL − RPMI 8226 myeloma − SK HEP-1 hepatoma − U937 histiocyticlymphoma − WERI-Rb-1 retinoblastoma + Y79.1 retinoblastoma + NT-2teratocarcinoma +

[0086] Activation of Y79.1 cells with PMA was found to increase theexpression of AC133 antigen (shown in FIG. 2). However, PMA activationof several other cell lines, or PHA activation of human PBMC was unableto induce the expression of AC133 antigen (data are shown in Table 2).AC133 antigen expression was not detectable on any of the CD34⁺ celllines tested. This finding, along with the lack of CD34 expression onthe Y79.1 cell line (shown in FIG. 3), excludes the possibility thatAC133 is directed to the CD34 antigen. AC133 antigen expression islimited to primitive stem and progenitor cells, unlike the CD34 antigen,which is also expressed on endothelium and fibroblasts (Krause et al.(1996) Blood 87:1-13).

[0087] The AC133 antigen is expressed on the CD34^(bright) population ofhuman progenitor cells isolated from fetal and adult bone marrow, fetalliver, cord blood, eukaphoresis (LP) packs and LP packs from cytokinemobilized donors. Typically it stains 30-50% of all CD34⁺ cells in thesepopulations. TABLE 2 Activation of Cell Lines Addition of PMA at 1 ng/mlfor: 0 hr 24 hr 48 hr 72 hr 96 hr 144 hr Y79.1 dim+ + + + + + KG1a − − −− − N/A K562 − − − − − N/A HEL 92.1.7 − − − − N/A N/A Jurkat − − − N/AN/A N/A 8402 − N/A N/A − N/A N/A Addition of PWM at 10 μg/ml for: 0 hr24 hr 48 hr Y79.1 dim+ + + KG1a − − −

[0088] Phenotyping of AC133 Positive Cells.

[0089] Phenotyping of AC133 and CD34 double positive cells wasaccomplished using 2 and 3 color FACS analysis, employing a panel ofconjugated antibodies directed to cell surface structures known to beexpressed on progenitor cells. Fetal liver, fetal and adult bone marrow,cord blood and peripheral blood were all used to determine the precisephenotype of AC133 positive cells. AC133 cells found in all of thesetissues are CD34^(bright), CD38^(−/+) and HLA-DR^(−/+). The data areshown in FIG. 4. The CD90 (Thy1)⁺ and CD117 (c-kit)⁺ stem cellpopulations are included within the AC133 positive population, as shownin FIG. 5. In a series of experiments performed with AC133immunomagnetically purified fetal liver cells, CD38-FITC conjugatedantibody stained 74.5% of the AC133 purified cells, while 24.8% wereCD38 negative. As expected, HLA-DR stained the majority of the cells(81.14%). CD90 is shown to stain 27.4% of the test cells, while CD117stained 90%. It is generally believed that primitive (repopulating)hematopoietic stem cells have the phenotype of CD34^(bright),CD38^(dim/neg), HLA-DR⁺, CD117^(dim) and CD90⁺. Thus, the AC133 antibodyrecognizes a phenotypically important population of human hematopoieticprogenitor cells.

[0090] Immunoprecipitation of the AC133 Antigen.

[0091] Immunoprecipiation experiments showed that the AC133 antigen hasa molecular weight of 120 kD. Biotin (Pierce) labeled, activated Y79.1and Weri-RB-1 cells were solubilized with lysis buffer: 2.5% Brij(Sigma), 25 mM Tris-HCl, pH 8.0, 125 mM NaCl, 2.5 mM EDTA, 2.2 Tg/mlAprotinin (Sigma) and 1 mM PMSF (Sigma). The lysates were incubated withAC133 and control antibodies after preclearing. Immunocomplexes werecollected on Staphylococcus aureus cells (CalBiochem) and heated forfive minutes at 95° C. in SDS-PAGE sample buffer with 1%2-mercaptoethanol. Immunoprecipitates were resolved by SDS-PAGE andtransferred to nitrocellulose membranes (Novex). Visualization wasaccomplished using streptavidin linked to horseradish peroxidase (HRP)(Amersham) and the Supersignal CL-HRP substrate system (Pierce). CD49d,CD71 and CD98 were used as controls, and their expected bands of 133 kD,92 kD and 80/40 kD were observed in the corresponding lanes. Theimmunoprecipitation with AC133 showed a distinct band corresponding to amolecular weight of 120 kD. This band was absent in the samples thatwere immunoprecipitated with the anti-CD34 antibodies AC101, HPCA1 andHPCA2, indicating that CD34 is not expressed in the Y79.1 cell line.This is consistent with the FACS data.

[0092] The data from a further experiment is shown in FIG. 6, wherebiotin labeled Y79.1, as well as KG1a cells, confirmed the AC133molecular weight data by comparing CD34 and AC133 precipitates on thesame gel. In this experiment, CD34 and Y79.1 antigens were precipitatedfrom biotinylated KG1a (CD34⁺) and Y79.1 lysates in adjacent lanes. Theresults clearly demonstrate that 1) each antibody precipitates its owndistinct antigen, and 2) that the molecular weight of these two antigensis distinctly different, being 110 and 127 kD, respectively. In controllanes 6 and 8, HPCA2 and 16D11 (anti-CD34) precipitate a band of 110 kDfrom KG1a lysate, but do not precipitate anything from Y79.1 lysate(lanes 7 and 9). AC133 precipitates a 120 kD protein from the Y79.1lysate (lane 10), but nothing from the KG1a lysate in lane 11. In lane12, KG1a and Y79.1 lysates were mixed, and AC133 Ag and CD34 wereco-precipitated. The results show that the two antigens are of differentmolecular weights.

[0093] AG133 magnetic bead conjugation. Purified AC133 antibody wasconjugated to magnetic amino-dextran beads using a standard protocol for4-(N-maleimidomethyl)-cyclohexane-1-carboxylic acid N-hydroxysuccinimideester (SMCC). AC133 antibody was added to SMCC activated beads at 5 Tgper OD₄₅₀ unit, and incubated at room temperature for two hours. Thereaction was stopped by the addition of θ-mercaptoethanol and NEM. Theconjugate was purified over two columns in the presence of a magneticfield, and eluted. The concentration was adjusted to OD₄₅₀=10, and OPGwas added for stabilization. The conjugate in PBS and 0.1% sodium azidewas filtered through a 0.2 Tm filter, and stored at 4° C.

[0094] Separation of Human Hematopoietic Progenitor Cells with AC133Magnetic Bead Conjugate.

[0095] AC133 direct magnetic bead conjugates were prepared and tested onbuffy coat PBMCs, fetal liver WC1, fetal bone marrow and adult bonemarrow. FIG. 8 shows the FACS dot plot of fetal liver cells purifiedwith AC133 bead conjugate using the miniMACS system and stained withglycophorin A-FITC and HCPA2-PE. The starting material contained 7.4%CD34⁺ cells, following AC133 purification, greater than 90% of AC133purified cells were bright CD34+. FIG. 9 shows that AC133 magneticconjugate was also very effective in enriching CD34⁺ cells from a buffycoat which contained about 0.26% CD34⁺ cells. The final purifiedpopulation was 64% positive for CD34, as shown by HPCA2-PE staining.This ability to separate cells in a magnetic purification system enablesfurther study of the functional and phenotypic properties of AC133.

[0096] Clonogenic Potential of AC133 Positive Cells.

[0097] AC133 magnetic beads selected cells purified from leukaphoresispacks were tested in clonogenicity assays using a commercially availablekit (Stem Cell Technologies, Vancouver, B.C.). By providing a controlledgrowth environment utilizing recombinant human growth factors thisculture assay identifies the major colony forming units (CFU) within aCD34 positive cell population. It provides information on thecomposition of progenitor cell populations, with respect to the relativepercentages of cells committed to a particular lineage specificdifferentiation. Typically in peripheral blood derived CD34⁺ cellpopulations BFU-e (burst forming units-erythroid), and CFU-GM (colonyforming units-granulocyte macrophage) are the predominant coloniesrecognized, being present at a 3:1 ratio. FIG. 10 shows the results froma typical clonogenicity experiment comparing AC133 and CD34 purifiedcells obtained from a split leukaphresis pack. Colonies obtained withunfractionated control cells are typically predominantly BFU-E (29.34%),with a smaller number of CFU-GM (5.14%). CD34 purified cells show asimilar distribution with 23.3% BFU-E, and 5.58% CFU-GM. In contrast,AC133 purified cells show a different pattern, with 13.1% BFU-E and10.2% CFU-GM. Calculations show that 58% of CFU-GM were recovered in theAC133 purified fraction, while only 13% of BFUEs were recovered.

[0098]FIG. 11 shows the results from a similar clonogenicity assayobtained following AC133 immunomagnetic purification. In thisexperiment, AC133 cells were positively selected, and then CD34 positivecells were positively selected from the AC133 negative flow-through.This design allowed the direct comparison of AC133⁺ cells with CD34⁺ butAC133⁻ cells from the same donor. The results indicate that 93.8% of theCFU-GM progenitors were recovered in the AC133 positive fraction, theremaining 6.2% being recovered from the CD34+/AC133⁻ fraction.Conversely, the CD34⁺AC133⁻ fraction contained 78.0% of the BFU-eprogenitors, while the remaining 22.0% were contained in the AC133⁺fraction.

[0099] The above experimental results rule out the possibility that ananti-AC133 antibody is an antibody to Fc receptors, or that ananti-AC133 antibody binds to stem cells via Fc receptor uptake. Furtherexperiments rule out the possibility that AC133 antibody staining is dueto free PE. AC133 antibody does not behave like an antibody to RTK, areceptor tyrosine kinase, TIE, a tyrosine kinase that containsimmunoglobulin-like domains and growth factor homology domains and whichis expressed in vascular endothelial cells and hematopoietic cells.AC133 antibody also does not behave like an antibody to P-glycoprotein,a 170 kD multi-drug resistance product which is also expressed inhematopoietic cells.

[0100] We have shown that AC133 antibody recognizes an antigen expressedonly on bright CD34+ cells in bone marrow, fetal liver and peripheralblood. This antibody and its antigen do not match the molecular weightor distribution of any known CD antigen. Apart from stem cells, AC133antibody has been shown to react with a human retinoblastoma cell linethat is negative for CD34 expression. AC133 antigen is, in addition, notexpressed on a number of CD34+ cell lines.

[0101] It is evident from the above results that the subject inventionprovides for a novel antigen found on primitive stem cells and a subsetof hematopoietic progenitor cells, as well as antibodies thatspecifically bind to the antigen. Expression of the antigen is highlytissue specific. It is only detected on a subset of hematopoieticprogenitor cells, and is present on substantially all cells that areactive in the CFU-GM assay. This highly specific distribution of AC133antigen makes it exceptionally useful as a reagent for isolating andcharacterizing human hematopoietic progenitor and stem cells.

[0102] Purification and Characterization of the AC133 Antigen.

[0103] The purification and characterization of the AC133 antigen, aswell as the isolation of a cDNA clone is described here. Protein andnucleic acid sequence analysis of this molecule indicate that the AC133antigen is the first described member of a new class of transmembranereceptors, having 5 transmembrane domains with little if any homology toknown G-protein coupled 7 transmembrane family members.

[0104] Antibody AC133 was prepared and purified as described above andconjugated to CNBr activated sepharose. CNBr activated sepharose waspurchased from Pharmacia (Alameda, Calif.), and mAb AC133 affinity resinwas prepared per the manufacturer's procedure using a 25 minute ligandcoupling reaction. The COS-7 and the WERI-Rb-1 retinoblastoma cell lineswere obtained from American Type Culture Collection (Rockville, Md.).Custom primers were synthesized by Operon Technologies (Alameda,Calif.).

[0105] Purification of the AC133 Antigen.

[0106] The AC133 antigen was isolated from 96 hour PMA activated Y79retinoblastoma cells (commercially available, for example, from ATCC).Cells (2×10⁹) were washed with PBS and lysed in 0.125M NaCl, 25mM TrispH 8, 0.005% NaN₃, 2.5mM EDTA, and 2.5% Brij 99/96 (2:1) detergentcontaining 1.0mM phenylmethyl sulfonylfluoride (PMSF) and a {fraction(1/1000)} dilution of a 2.2 mg/ml solution of aprotinin containing 4.1trypsin inhibitor units per mg (Sigma). Cells were vortexedintermittently for 5 minutes at room temperature and then left on icefor 20 minutes. Cell nuclei and debris were removed by centrifugation at12,000×G for 10 minutes. Lysate supernatant was filtered through a 0.2μM filter prior to loading onto 0.5 mL mAb AC133 affinity columnequilibrated in wash buffer (0.125 M NaCl, 25 mM Tris pH 8.0, 0.01%NaN₃, 2.5 mM EDTA, 0.1% Brij). The column was washed extensively withwash buffer and the antigen was eluted in 50 mM ethanolamine pH 11.5,0.1% Brij, 0.01% NaN₃. The pH was immediately adjusted to neutral withHCl. Passage of the antigen eluate over a 300 μl bed volume DEAE columnequilibrated in wash buffer removed many of contaminating proteins, anda second affinity chromatography step using an AC133 antibody column asdescribed above resulted in >95% pure AC133 antigen amenable toproteolysis and protein sequence analysis. The purity and identity ofAC133 antigen was confirmed by sodium dodecyl sulfate-polyacrylamide gelelectrophoresis (SDS-PAGE) and Western analysis (Towbin, H., T.Staehelin, and J. Gordon (1979) PNAS 76,4350-4354; Towbin, H. and J.Gordon (1984) J. Immunol. Meth 72:313-340).

[0107] Endoglyconase Treatment of the Purified AC133 antigen.

[0108] One microgram of AC133 antigen was resuspended in 50 μl water and125 μl 0.1 M 2-mercaptoethanol and 0.5% SDS. The protein was denaturedat 100° C. for 5 minutes. Denatured mixture (35 μl) was added to each of5 tubes, together with 25 μl 0.5 M Tris pH 8, 10 μl water, 10 μl 10%NP-40. 0-0.1 unit PNGase F (Sigma) was added to each tube, and the tubeswere incubated at 30° C. overnight. Deglycosylated antigen wasvisualized on a silver stained SDS-polyacrylamide gel.

[0109] Lysyl Endopeptidase Digestion of the AC133 Antigen and Isolationof Peptides.

[0110] AC133 antigen was precipitated from 1.4 mL of 2 μg/mL affinitycolumn eluate by the addition of TCA to 10%. The precipitated dryprotein was suspended in 25 μL of solution digest buffer (8M urea, 400mM Tris pH 7.8), to which 5 μl of 45 mM DTT was added and the mixtureincubated at 50° C. for 15 min. After cooling to room temperature, 5 μlof 100 mM iodoacetamide was added and the mix was incubated for anadditional 15 minutes. Distilled water (70 μl) was added, diluting theurea to 2 M, and 2 pmol of the lysyl endopeptidase, LysC (commerciallyavailable from Wako Chemicals, USA), was added. The digestion wascarried out at 37° C. for 24 hours. Peptides were isolated by HPLCseparation on a VYDAC narrowbore C18 reverse phase column with a 4-32%acetonitrile gradient in 0.1% trifluoroacetic acid (TFA).

[0111] Protein Sequence Analysis of AC133 Antigen Peptides.

[0112] N-terminal sequence analysis was determined using Edman chemistry(Edman, P., Begg, G. (1967) Eur. J. Biochem. 1, 80-91; Huwick, R. M.,Hunkapillar, M. W., Hood, L. E., and Dreyer, W. J. (1987) J. Biol Chem.256, p. 7990) on an Applied Biosystems 477A or 473A liquid pulse proteinsequenator. PTH-Amino acids were separated on a Brownlee C-18 reversephase column (2.1 mm×22 cm) at 55° C. in buffer A (3.5% tetrahydrofuranwith addition of 2 to 4% ABI Premix Buffer concentrate from AppliedBiosystems to buffer B (acetonitrile), with a 12-36% buffer B lineargradient over 18 min, followed by a 13 min. isocratic period at 36% B.

[0113] Isolation and Protein Sequencing of the AC133 Antigen.

[0114] The 120 kD AC133 antigen was isolated by immunoaffinitychromatography from a retinoblastoma cell line, Y79, which was PMAactivated for 96 hours prior to harvest. Sequential affinitychromatography and DEAE chromatography were utilized to generate >95%pure AC133 antigen by SDS-PAGE and silver staining and the identity ofthe purified molecule as the AC133 antigen was confirmed by Westernblotting. De-glycosylation of the antigen with PGNase F to removeN-linked sugar shows that approximately 30 kD of the molecular weight isdue to glycosylation. Repeated initial attempts to sequence theN-terminus of the AC133 antigen failed, suggesting that this protein isamino-terminally blocked. However, digestion of the purified antigenwith lysyl endopeptidase followed by reverse phase HPLC, yielded fourpeptide sequences with lengths of 12-16 amino acids. Searches of themajor protein and nucleic acid databases with the peptide and resultingdegenerate oligonucleotide sequences indicated that the AC133 antigencould not be identified with any described molecules. (The amino acidsequence has now been deduced from cDNA cloning and is shown in FIG.12.)

[0115] cDNA Cloning.

[0116] Total RNA was isolated from WERI-Rb-1 retinoblastoma cells(available from the American Type Culture Collection; Rockville, Md.)and poly A⁺ RNA was prepared using the Poly A⁺ Tract System (PromegaCorp., Madison, Wis.). cDNA was synthesized (Guebler, U. and B. J.Hoffman (1983) Gene 25:263) using superscript reverse transcriptase(GIBCO BRL, Gaithersburg, Md.) and an oligo dT primer. The blunted cDNAwas ligated to nonself-complimentary Bst XI adaptors and gel purified toremove unligated adaptors and small fragments. The Tinkered cDNA wasthen ligated into the pcDNA-1 expression vector (Invitrogen, San Diego,Calif.) and electroporated into Escherichia coli strain MC1061/P3(Dower, W. J. (1990) Genetic Engineering V. 12 Edited by J. K. Seflow,Plenum Press, New York 275-295. (Electroporation of Bacteria: a generalapproach to genetic transformation); Ausubel, F. M., R. Brent, R. E.Kingston, D. D. Moore, J. G. Seidman, J. A. Smith and K. Struhl.1987-1994 Current Protocols in Molecular Biology. John Wilest Sons;N.Y.). WERI-Rb-1 library cDNA (100 ng/reaction) was used as a PCRtemplate with 100 pmol each degenerate sense and antisense primersdesigned from the protein sequence of four AC133 antigen peptides. PCRreactions were carried out in buffer (50 mM KCl, 10 mM Tris pH 9, 0.1%Triton X-100, 1.5 mM MgCl, 0.2 mM (each) dNTP's) with 5 units of Taq DNApolymerase per reaction (Promega Corp, Madison, Wis.). Amplification wascarried out in an MJ research (data) instrument as follows: 92° C. for 1min, 55-37° C. for 1 min, 72° C. for 3 min, 35 cycles. Afteramplification the reaction mixtures were run on 1% agarose gels, andunique bands not appearing in the individual primer controls were gelpurified and cloned into pCR 2.1 using a TA Cloning Kit (Invitrogen, SanDiego, Calif.). The 5′ and 3′ ends of the gene were isolated byhemi-specific PCR with nested sets of AC133 antigen gene specificprimers and library specific primers. Twenty cycles of single-strandedPCR were performed with each gene specific primer in a 50 μl reactionvolume with 100 ng of the library cDNA and 10 pmol each primer in PCRreaction buffer (described above) with 5 units of Taq polymerase. Analiquot (10 μl) of this reaction mix was removed and used as templatefor a second, 35 cycle, PCR reaction using both the gene specific primerand the library specific primer. An aliquot (5 μl) of this PCR reactionmix was then used for another 35 cycles of reaction using nested libraryand gene specific primers. Bands corresponding to the 5′ and 3′ ends ofthe gene were gel purified and cloned into pCR 2.1. Overlapping cDNAclones were sequenced by the dideoxy chain reaction using fluorescentdye terminators and an ABI sequencer (Applied Biosystems, Foster City,Calif.)

[0117] Isolation of a cDNA Clone of the AC133 Antigen.

[0118] To isolate the cDNA for this protein, a cDNA library was preparedfrom the WERI-Rb-1 retinoblastoma cell line that expresses approximately10-fold more AC133 antigen than PMA activated Y79 cells. Degenerateprimers were used in low stringency PCR reactions with the library toyield a 1.7 kb fragment that contained the correct sequence of peptide 3at the 5′ end and the correct sequence of peptide 4 at the 3 prime end.Additionally, the sequence of peptide 2 was found within the fragment inthe correct reading frame. Hemi-specific PCR with gene specific primersand library specific primers yielded additional 1.2 kB and 2 kBfragments corresponding to the 5′ and 3′ ends of the gene andoverlapping with the initial 1.7 kB clone.

[0119] Sequencing of the three partial clones yielded a 4 kB cDNAcontaining an open reading frame of 3.0 kB, but also containing a 128 bpintron that appears to be associated with the poly A⁺ version of thegene, and does not contain eukaryotic consensus splice sequences. Toisolate an intact stem cell derived clone without the intron, AC133⁺stem cells were isolated from fetal liver utilizing a magnetic conjugateof mAb AC133 and the Miltenyi magnetic separation system (MiltenyiBiotech, GMBH). Total RNA was isolated from these cells, and used as atemplate for RT-PCR reactions. Primers designed to span the introngenerate a single 582 bp fragment with the poly A+ derived cDNAtemplate, but generate a single 454 bp fragment without the intron fromtotal RNA in AC133⁺ cell lines (FIG. 3), suggesting that the splicedmRNA is the major product within the total RNA pool. RT-PCR was utilizedto generate cDNA clones originating before the start methionine andcontaining the complete cDNA sequence. The full length cDNA encodingAC133 antigen predicts a protein of 863 amino acids with a molecularweight of 96.8 kD (FIG. 4). Hydrophobicity analysis of the sequence(FIG. 5) and transmembrane helix algorithms indicate that the proteinspans the cell membrane a total of five times (FIG. 6) predicting thepresence of two large (255 and 280 amino acids) extracellular loops anda C-terminal cytoplasmic tail. Other structural features suggested bythe protein sequence include leucine zipper motifs in both of theputative large extracellular loops and six consensus sequences forN-glycosylation.

[0120] Expression of the AC133 Antigen in Transfected COS-7 Cells.

[0121] AC133 positive cells (1×10⁷) were isolated from fetal liver asdescribed above. Total RNA was isolated using RNAzol (Gibco BRL,Gaithersburg, Md.) as described (Chomczynski, P. and Sacchi, N. (1987)Anal. Biochem. 162, 156). RT-PCR was performed using the Promega AccessRT-PCR system (Promega Corp, Madison, Wis.) with 10 ng total RNAtemplate and primers directed before the start methionine and after thestop codon. The 2.8 kb band corresponding to the coding region of thegene was cloned into the Invitrogen directional eukaryotic TA cloningvector (pCR 3.1) containing the CMV promoter. Subconfluent COS-7 cells(available from the ATCC, Rockville, Md.) were transfected with 5 μg ofcloned DNA by electroporation and incubated for 48 hours prior to FACSanalysis. Transfected COS-7 cells were stained with 50 ng/100 μl testmAb AC133-PE, and analyzed with a Becton Dickenson (San Jose, Calif.)FACS scan.

[0122] Expression of the AC133 Antigen in COS Cells.

[0123] COS cells transfected with the AC133 antigen gene were stainedwith mAb AC133-PE and analyzed by FACS (FIG. 7). Cos cells transfectedwith the AC133 antigen gene stain brightly with mAB AC133-PE, however,untransfected cells, cells transfected with empty vector or the gene forCD-8 do not stain with this antibody.

[0124] AC133 Expression in Various Lymphoid and Non-lymphoid Cell Lines.

[0125] The presence of AC133 antigen transcript in a variety of celllines was assessed by Northern analysis. Northern blot analysis wasperformed by using Clontech (Palo Alto, Calif.) multiple tissue northernblots, and by resolving RNA samples on a 1% agarose-2M formaldehyde geland capillary blotting overnight into nylon membrane. Total RNA wasisolated with Tri Reagent, and 15 μg was loaded per lane. Staining ofthe blot with methylene blue was used to monitor RNA concentrations. An800 bp EcoRI fragment of the cDNA was labelled with 32P-dCTP by randompriming and used as a probe.

[0126] The presence of AC133 antigen transcript in a variety of celllines was assessed by Northern analysis. A 4.4kB MRNA transcript wasdetectable in WERI-Rb-1 cells as well as Y79 cells and MACS-isolatedAC133+ fetal liver cells. While expression of the AC133 antigen isenhanced in Y79 cells upon PMA activation, the corresponding MRNAappears to be downregulated. In normal hemtopoetic tissue, the AC133antigen message is detectable in fetal liver, and weakly detectable inadult bone marrow as expected due to the fact that AC133+ cells in thesetissues are in a minority. The AC133 antigen transcript was 10 alsonoted in non-lymphoid tissues, particularly in pancreas, kidney, andplacenta. Weaker signals were observed for liver, lung, brain, andheart. This is in contrast to immunohistochemical staining of paraffintissue sections were AC133 antigen expression was detectable only inbone marrow.

[0127] In a similar manner, other antibodies have been developed thatare specific for the AC133 antigen. The following table showsantibodies, immunogens, isotypes, and cross blocking for a panel of suchantibodies. AC 133 cross Antibody Immunogen Isotype blocking AC133 HSCIgG1 kappa +++ AC139 WERI-Rb-1 IgG1 kappa +++ AC140 WERI-Rb-1 IgG1 kappa+/− AC141 WERI-Rb-1 IgG1 kappa − AC142 WERI-Rb-1 IgG1 kappa ND

[0128] All publications and patent applications cited in thisspecification are herein incorporated by reference as if each individualpublication or patent application were specifically and individuallyindicated to be incorporated by reference.

[0129] Although the foregoing invention has been described in somedetail byway of illustration and example for purposes of clarity ofunderstanding, it will be readily apparent to those of ordinary skill inthe art in light of the teachings of this invention that certain changesand modifications can be made thereto without departing from the spiritor scope of the appended claims.

1 2 1 3804 DNA Homo sapiens CDS (38)..(2632) 1 ccaagttcta cctcatgtttggaggatctt gctagct atg gcc ctc gta ctc ggc 55 Met Ala Leu Val Leu Gly 15 tcc ctg ttg ctg ctg ggg ctg tgc ggg aac tcc ttt tca gga ggg cag 103Ser Leu Leu Leu Leu Gly Leu Cys Gly Asn Ser Phe Ser Gly Gly Gln 10 15 20cct tca tcc aca gat gct cct aag gct tgg aat tat gaa ttg cct gca 151 ProSer Ser Thr Asp Ala Pro Lys Ala Trp Asn Tyr Glu Leu Pro Ala 25 30 35 acaaat tat gag acc caa gac tcc cat aaa gct gga ccc att ggc att 199 Thr AsnTyr Glu Thr Gln Asp Ser His Lys Ala Gly Pro Ile Gly Ile 40 45 50 ctc tttgaa cta gtg cat atc ttt ctc tat gtg gta cag ccg cgt gat 247 Leu Phe GluLeu Val His Ile Phe Leu Tyr Val Val Gln Pro Arg Asp 55 60 65 70 ttc ccagaa gat act ttg aga aaa ttc tta cag aag gca tat gaa tcc 295 Phe Pro GluAsp Thr Leu Arg Lys Phe Leu Gln Lys Ala Tyr Glu Ser 75 80 85 aaa att gattat gac aag cca gaa act gta atc tta ggt cta aag att 343 Lys Ile Asp TyrAsp Lys Pro Glu Thr Val Ile Leu Gly Leu Lys Ile 90 95 100 gtc tac tatgaa gca ggg att att cta tgc tgt gtc ctg ggg ctg ctg 391 Val Tyr Tyr GluAla Gly Ile Ile Leu Cys Cys Val Leu Gly Leu Leu 105 110 115 ttt att attctg atg cct ctg gtg ggg tat ttc ttt tgt atg tgt cgt 439 Phe Ile Ile LeuMet Pro Leu Val Gly Tyr Phe Phe Cys Met Cys Arg 120 125 130 tgc tgt aacaaa tgt ggt gga gaa atg cac cag cga cag aag gaa aat 487 Cys Cys Asn LysCys Gly Gly Glu Met His Gln Arg Gln Lys Glu Asn 135 140 145 150 ggg cccttc ctg agg aaa tgc ttt gca atc tcc ctg ttg gtg att tgt 535 Gly Pro PheLeu Arg Lys Cys Phe Ala Ile Ser Leu Leu Val Ile Cys 155 160 165 ata ataata agc att ggc atc ttc tat ggt ttt gtg gca aat cac cag 583 Ile Ile IleSer Ile Gly Ile Phe Tyr Gly Phe Val Ala Asn His Gln 170 175 180 gta agaacc cgg atc aaa agg agt cgg aaa ctg gca gat agc aat ttc 631 Val Arg ThrArg Ile Lys Arg Ser Arg Lys Leu Ala Asp Ser Asn Phe 185 190 195 aag gacttg cga act ctc ttg aat gaa act cca gag caa atc aaa tat 679 Lys Asp LeuArg Thr Leu Leu Asn Glu Thr Pro Glu Gln Ile Lys Tyr 200 205 210 ata ttggcc cag tac aac act acc aag gac aag gcg ttc aca gat ctg 727 Ile Leu AlaGln Tyr Asn Thr Thr Lys Asp Lys Ala Phe Thr Asp Leu 215 220 225 230 aacagt atc aat tca gtg cta gga ggc gga att ctt gac cga ctg aga 775 Asn SerIle Asn Ser Val Leu Gly Gly Gly Ile Leu Asp Arg Leu Arg 235 240 245 cccaac atc atc cct gtt ctt gat gag att aag tcc atg gca aca gcg 823 Pro AsnIle Ile Pro Val Leu Asp Glu Ile Lys Ser Met Ala Thr Ala 250 255 260 atcaag gag acc aaa gag gcg ttg gag aac atg aac agc acc ttg aag 871 Ile LysGlu Thr Lys Glu Ala Leu Glu Asn Met Asn Ser Thr Leu Lys 265 270 275 agcttg cac caa caa agt aca cag ctt agc agc agt ctg acc agc gtg 919 Ser LeuHis Gln Gln Ser Thr Gln Leu Ser Ser Ser Leu Thr Ser Val 280 285 290 aaaact agc ctg cgg tca tct ctc aat gac cct ctg tgc ttg gtg cat 967 Lys ThrSer Leu Arg Ser Ser Leu Asn Asp Pro Leu Cys Leu Val His 295 300 305 310cca tca agt gaa acc tgc aac agc atc aga ttg tct cta agc cag ctg 1015 ProSer Ser Glu Thr Cys Asn Ser Ile Arg Leu Ser Leu Ser Gln Leu 315 320 325aat agc aac cct gaa ctg agg cag ctt cca ccc gtg gat gca gaa ctt 1063 AsnSer Asn Pro Glu Leu Arg Gln Leu Pro Pro Val Asp Ala Glu Leu 330 335 340gac aac gtt aat aac gtt ctt agg aca gat ttg gat ggc ctg gtc caa 1111 AspAsn Val Asn Asn Val Leu Arg Thr Asp Leu Asp Gly Leu Val Gln 345 350 355cag ggc tat caa tcc ctt aat gat ata cct gac aga gta caa cgc caa 1159 GlnGly Tyr Gln Ser Leu Asn Asp Ile Pro Asp Arg Val Gln Arg Gln 360 365 370acc acg act gtc gta gca ggt atc aaa agg gtc ttg aat tcc att ggt 1207 ThrThr Thr Val Val Ala Gly Ile Lys Arg Val Leu Asn Ser Ile Gly 375 380 385390 tca gat atc gac aat gta act cag cgt ctt cct att cag gat ata ctc 1255Ser Asp Ile Asp Asn Val Thr Gln Arg Leu Pro Ile Gln Asp Ile Leu 395 400405 tca gca ttc tct gtt tat gtt aat aac act gaa agt tac atc cac aga 1303Ser Ala Phe Ser Val Tyr Val Asn Asn Thr Glu Ser Tyr Ile His Arg 410 415420 aat tta cct aca ttg gaa gag tat gat tca tac tgg tgg ctg ggt ggc 1351Asn Leu Pro Thr Leu Glu Glu Tyr Asp Ser Tyr Trp Trp Leu Gly Gly 425 430435 ctg gtc atc tgc tct ctg ctg acc ctc atc gtg att ttt tac tac ctg 1399Leu Val Ile Cys Ser Leu Leu Thr Leu Ile Val Ile Phe Tyr Tyr Leu 440 445450 ggc tta ctg tgt ggc gtg tgc ggc tat gac agg cat gcc acc ccg acc 1447Gly Leu Leu Cys Gly Val Cys Gly Tyr Asp Arg His Ala Thr Pro Thr 455 460465 470 acc cga ggc tgt gtc tcc aac acc gga ggc gtc ttc ctc atg gtt gga1495 Thr Arg Gly Cys Val Ser Asn Thr Gly Gly Val Phe Leu Met Val Gly 475480 485 gtt gga tta agt ttc ctc ttt tgc tgg ata ttg atg atc att gtg gtt1543 Val Gly Leu Ser Phe Leu Phe Cys Trp Ile Leu Met Ile Ile Val Val 490495 500 ctt acc ttt gtc ttt ggt gca aat gtg gaa aaa ctg atc tgt gaa cct1591 Leu Thr Phe Val Phe Gly Ala Asn Val Glu Lys Leu Ile Cys Glu Pro 505510 515 tac acg agc aag gaa tta ttc cgg gtt ttg gat aca ccc tac tta cta1639 Tyr Thr Ser Lys Glu Leu Phe Arg Val Leu Asp Thr Pro Tyr Leu Leu 520525 530 aat gaa gac tgg gaa tac tat ctc tct ggg aag cta ttt aat aaa tca1687 Asn Glu Asp Trp Glu Tyr Tyr Leu Ser Gly Lys Leu Phe Asn Lys Ser 535540 545 550 aaa atg aag ctc act ttt gaa caa gtt tac agt gac tgc aaa aaaaat 1735 Lys Met Lys Leu Thr Phe Glu Gln Val Tyr Ser Asp Cys Lys Lys Asn555 560 565 aga ggc act tac ggc act ctt cac ctg cag aac agc ttc aat atcagt 1783 Arg Gly Thr Tyr Gly Thr Leu His Leu Gln Asn Ser Phe Asn Ile Ser570 575 580 gaa cat ctc aac att aat gag cat act gga agc ata agc agt gaattg 1831 Glu His Leu Asn Ile Asn Glu His Thr Gly Ser Ile Ser Ser Glu Leu585 590 595 gaa agt ctg aag gta aat ctt aat atc ttt ctg ttg ggt gca gcagga 1879 Glu Ser Leu Lys Val Asn Leu Asn Ile Phe Leu Leu Gly Ala Ala Gly600 605 610 aga aaa aac ctt cag gat ttt gct gct tgt gga ata gac aga atgaat 1927 Arg Lys Asn Leu Gln Asp Phe Ala Ala Cys Gly Ile Asp Arg Met Asn615 620 625 630 tat gac agc tac ttg gct cag act ggt aaa tcc ccc gca ggagtg aat 1975 Tyr Asp Ser Tyr Leu Ala Gln Thr Gly Lys Ser Pro Ala Gly ValAsn 635 640 645 ctt tta tca ttt gca tat gat cta gaa gca aaa gca aac agtttg ccc 2023 Leu Leu Ser Phe Ala Tyr Asp Leu Glu Ala Lys Ala Asn Ser LeuPro 650 655 660 cca gga aat ttg agg aac tcc ctg aaa aga gat gca caa actatt aaa 2071 Pro Gly Asn Leu Arg Asn Ser Leu Lys Arg Asp Ala Gln Thr IleLys 665 670 675 aca att cac cag caa cga gtc ctt cct ata gaa caa tca ctgagc act 2119 Thr Ile His Gln Gln Arg Val Leu Pro Ile Glu Gln Ser Leu SerThr 680 685 690 cta tac caa agc gtc aag ata ctt caa cgc aca ggg aat ggattg ttg 2167 Leu Tyr Gln Ser Val Lys Ile Leu Gln Arg Thr Gly Asn Gly LeuLeu 695 700 705 710 gag aga gta act agg att cta gct tct ctg gat ttt gctcag aac ttc 2215 Glu Arg Val Thr Arg Ile Leu Ala Ser Leu Asp Phe Ala GlnAsn Phe 715 720 725 atc aca aac aat act tcc tct gtt att att gag gaa actaag aag tat 2263 Ile Thr Asn Asn Thr Ser Ser Val Ile Ile Glu Glu Thr LysLys Tyr 730 735 740 ggg aga aca ata ata gga tat ttt gaa cat tat ctg cagtgg atc gag 2311 Gly Arg Thr Ile Ile Gly Tyr Phe Glu His Tyr Leu Gln TrpIle Glu 745 750 755 ttc tct atc agt gag aaa gtg gca tcg tgc aaa cct gtggcc acc gct 2359 Phe Ser Ile Ser Glu Lys Val Ala Ser Cys Lys Pro Val AlaThr Ala 760 765 770 cta gat act gct gtt gat gtc ttt ctg tgt agc tac attatc gac ccc 2407 Leu Asp Thr Ala Val Asp Val Phe Leu Cys Ser Tyr Ile IleAsp Pro 775 780 785 790 ttg aat ttg ttt tgg ttt ggc ata gga aaa gct actgta ttt tta ctt 2455 Leu Asn Leu Phe Trp Phe Gly Ile Gly Lys Ala Thr ValPhe Leu Leu 795 800 805 ccg gct cta att ttt gcg gta aaa ctg gct aag tactat cgt cga atg 2503 Pro Ala Leu Ile Phe Ala Val Lys Leu Ala Lys Tyr TyrArg Arg Met 810 815 820 gat tcg gag gac gtg tac gat gat gtt gaa act ataccc atg aaa aat 2551 Asp Ser Glu Asp Val Tyr Asp Asp Val Glu Thr Ile ProMet Lys Asn 825 830 835 atg gaa aat ggt aat aat ggt tat cat aaa gat catgta tat ggt att 2599 Met Glu Asn Gly Asn Asn Gly Tyr His Lys Asp His ValTyr Gly Ile 840 845 850 cac aat cct gtt atg aca agc cca tca caa cattgatagctga tgttgaaact 2652 His Asn Pro Val Met Thr Ser Pro Ser Gln His855 860 865 gcttgagcat caggatactc aaagtggaaa ggatcacaga tttttggtagtttctgggtc 2712 tacaaggact ttccaaatcc aggagcaacg ccagtggcaa cgtagtgactcaggcgggca 2772 ccaaggcaac ggcaccattg gtctctgggt agtgctttaa gaatgaacacaatcacgtta 2832 tagtccatgg tccatcacta ttcaaggatg actccctccc ttcctgtctatttttgtttt 2892 ttactttttt acactgagtt tctatttaga cactacaaca tatggggtgtttgttcccat 2952 tggatgcatt tctatcaaaa ctctatcaaa tgtgatggct agattctaacatattgccat 3012 gtgtggagtg tgctgaacac acaccagttt acaggaaaga tgcattttgtgtacagtaaa 3072 cggtgtatat accttttgtt accacagagt tttttaaaca aatgagtattataggacttt 3132 cttctaaatg agctaaataa gtcaccattg acttcttggt gctgttgaaaataatccatt 3192 ttcactaaaa gtgtgtgaaa cctacagcat attcttcacg cagagattttcatctattat 3252 actttatcaa agattggcca tgttccactt ggaaatggca tgcaaaagccatcatagaga 3312 aacctgcgta actccatctg acaaattcaa aagagagaga gagatcttgagagagaaatg 3372 ctgttcgttc aaaagtggag ttgttttaac agatgccaat tacggtgtacagtttaacag 3432 agttttctgt tgcattagga taaacattaa ttggagtgca gctaacatgagtatcatcag 3492 actagtatca agtgttctaa aatgaaatat gagaagatcc tgtcacaattcttagatctg 3552 gtgtccagca tggatgaaac ctttgagttt ggtccctaaa tttgcatgaaagcacaaggt 3612 aaatattcat ttgcttcagg agtttcatgt tggatctgtc attatcaaaagtgatcagca 3672 atgaagaact ggtcggacaa aatttaacgt tgatgtaatg gaattccagatgtaggcatt 3732 ccccccaggt cttttcatgt gcagattgca gttctgattc atttgaataaaaaggaactt 3792 ggaaaaaaaa aa 3804 2 865 PRT Homo sapiens 2 Met Ala LeuVal Leu Gly Ser Leu Leu Leu Leu Gly Leu Cys Gly Asn 1 5 10 15 Ser PheSer Gly Gly Gln Pro Ser Ser Thr Asp Ala Pro Lys Ala Trp 20 25 30 Asn TyrGlu Leu Pro Ala Thr Asn Tyr Glu Thr Gln Asp Ser His Lys 35 40 45 Ala GlyPro Ile Gly Ile Leu Phe Glu Leu Val His Ile Phe Leu Tyr 50 55 60 Val ValGln Pro Arg Asp Phe Pro Glu Asp Thr Leu Arg Lys Phe Leu 65 70 75 80 GlnLys Ala Tyr Glu Ser Lys Ile Asp Tyr Asp Lys Pro Glu Thr Val 85 90 95 IleLeu Gly Leu Lys Ile Val Tyr Tyr Glu Ala Gly Ile Ile Leu Cys 100 105 110Cys Val Leu Gly Leu Leu Phe Ile Ile Leu Met Pro Leu Val Gly Tyr 115 120125 Phe Phe Cys Met Cys Arg Cys Cys Asn Lys Cys Gly Gly Glu Met His 130135 140 Gln Arg Gln Lys Glu Asn Gly Pro Phe Leu Arg Lys Cys Phe Ala Ile145 150 155 160 Ser Leu Leu Val Ile Cys Ile Ile Ile Ser Ile Gly Ile PheTyr Gly 165 170 175 Phe Val Ala Asn His Gln Val Arg Thr Arg Ile Lys ArgSer Arg Lys 180 185 190 Leu Ala Asp Ser Asn Phe Lys Asp Leu Arg Thr LeuLeu Asn Glu Thr 195 200 205 Pro Glu Gln Ile Lys Tyr Ile Leu Ala Gln TyrAsn Thr Thr Lys Asp 210 215 220 Lys Ala Phe Thr Asp Leu Asn Ser Ile AsnSer Val Leu Gly Gly Gly 225 230 235 240 Ile Leu Asp Arg Leu Arg Pro AsnIle Ile Pro Val Leu Asp Glu Ile 245 250 255 Lys Ser Met Ala Thr Ala IleLys Glu Thr Lys Glu Ala Leu Glu Asn 260 265 270 Met Asn Ser Thr Leu LysSer Leu His Gln Gln Ser Thr Gln Leu Ser 275 280 285 Ser Ser Leu Thr SerVal Lys Thr Ser Leu Arg Ser Ser Leu Asn Asp 290 295 300 Pro Leu Cys LeuVal His Pro Ser Ser Glu Thr Cys Asn Ser Ile Arg 305 310 315 320 Leu SerLeu Ser Gln Leu Asn Ser Asn Pro Glu Leu Arg Gln Leu Pro 325 330 335 ProVal Asp Ala Glu Leu Asp Asn Val Asn Asn Val Leu Arg Thr Asp 340 345 350Leu Asp Gly Leu Val Gln Gln Gly Tyr Gln Ser Leu Asn Asp Ile Pro 355 360365 Asp Arg Val Gln Arg Gln Thr Thr Thr Val Val Ala Gly Ile Lys Arg 370375 380 Val Leu Asn Ser Ile Gly Ser Asp Ile Asp Asn Val Thr Gln Arg Leu385 390 395 400 Pro Ile Gln Asp Ile Leu Ser Ala Phe Ser Val Tyr Val AsnAsn Thr 405 410 415 Glu Ser Tyr Ile His Arg Asn Leu Pro Thr Leu Glu GluTyr Asp Ser 420 425 430 Tyr Trp Trp Leu Gly Gly Leu Val Ile Cys Ser LeuLeu Thr Leu Ile 435 440 445 Val Ile Phe Tyr Tyr Leu Gly Leu Leu Cys GlyVal Cys Gly Tyr Asp 450 455 460 Arg His Ala Thr Pro Thr Thr Arg Gly CysVal Ser Asn Thr Gly Gly 465 470 475 480 Val Phe Leu Met Val Gly Val GlyLeu Ser Phe Leu Phe Cys Trp Ile 485 490 495 Leu Met Ile Ile Val Val LeuThr Phe Val Phe Gly Ala Asn Val Glu 500 505 510 Lys Leu Ile Cys Glu ProTyr Thr Ser Lys Glu Leu Phe Arg Val Leu 515 520 525 Asp Thr Pro Tyr LeuLeu Asn Glu Asp Trp Glu Tyr Tyr Leu Ser Gly 530 535 540 Lys Leu Phe AsnLys Ser Lys Met Lys Leu Thr Phe Glu Gln Val Tyr 545 550 555 560 Ser AspCys Lys Lys Asn Arg Gly Thr Tyr Gly Thr Leu His Leu Gln 565 570 575 AsnSer Phe Asn Ile Ser Glu His Leu Asn Ile Asn Glu His Thr Gly 580 585 590Ser Ile Ser Ser Glu Leu Glu Ser Leu Lys Val Asn Leu Asn Ile Phe 595 600605 Leu Leu Gly Ala Ala Gly Arg Lys Asn Leu Gln Asp Phe Ala Ala Cys 610615 620 Gly Ile Asp Arg Met Asn Tyr Asp Ser Tyr Leu Ala Gln Thr Gly Lys625 630 635 640 Ser Pro Ala Gly Val Asn Leu Leu Ser Phe Ala Tyr Asp LeuGlu Ala 645 650 655 Lys Ala Asn Ser Leu Pro Pro Gly Asn Leu Arg Asn SerLeu Lys Arg 660 665 670 Asp Ala Gln Thr Ile Lys Thr Ile His Gln Gln ArgVal Leu Pro Ile 675 680 685 Glu Gln Ser Leu Ser Thr Leu Tyr Gln Ser ValLys Ile Leu Gln Arg 690 695 700 Thr Gly Asn Gly Leu Leu Glu Arg Val ThrArg Ile Leu Ala Ser Leu 705 710 715 720 Asp Phe Ala Gln Asn Phe Ile ThrAsn Asn Thr Ser Ser Val Ile Ile 725 730 735 Glu Glu Thr Lys Lys Tyr GlyArg Thr Ile Ile Gly Tyr Phe Glu His 740 745 750 Tyr Leu Gln Trp Ile GluPhe Ser Ile Ser Glu Lys Val Ala Ser Cys 755 760 765 Lys Pro Val Ala ThrAla Leu Asp Thr Ala Val Asp Val Phe Leu Cys 770 775 780 Ser Tyr Ile IleAsp Pro Leu Asn Leu Phe Trp Phe Gly Ile Gly Lys 785 790 795 800 Ala ThrVal Phe Leu Leu Pro Ala Leu Ile Phe Ala Val Lys Leu Ala 805 810 815 LysTyr Tyr Arg Arg Met Asp Ser Glu Asp Val Tyr Asp Asp Val Glu 820 825 830Thr Ile Pro Met Lys Asn Met Glu Asn Gly Asn Asn Gly Tyr His Lys 835 840845 Asp His Val Tyr Gly Ile His Asn Pro Val Met Thr Ser Pro Ser Gln 850855 860 His 865

What is claimed is:
 1. An antibody that specifically binds to AC133antigen.
 2. An antibody according to claim 1 , wherein said antibody isa monoclonal antibody produced by a hybridoma cell line.
 3. A monoclonalantibody according to claim 2 , wherein said antibody blockssimultaneous binding to said antigen by the antibody produced by thehybridoma cell line ATCC.
 4. A monoclonal antibody according to claim 1, wherein said antibody is induced through contralateral immunization.5. A monoclonal antibody according to claim 1 , produced by thehybridoma cell line ATCC ______.
 6. A method for enrichment ofhematopoietic stem or progenitor cells or both, said method comprising:combining a mixed population of human cells comprising hematopoieticstem or progenitor cells or both with a reagent that specifically bindsto the hematopoietic progenitor cell antigen recognized by the antibodyproduced by the hybridoma cell line ATCC ______; and selecting for thosecells that bind said reagent; wherein said selected cells are enrichedin hematopoietic stem or progenitor cell activity or both, depending onwhether said mixed popluation of human cells contained hematopoieticstem or progenitor cells or both, respectively.
 7. A method according toclaim 6 , further comprising: combining said mixed population of humancells with a reagent that specifically recognize at least one of thecell surface markers CD90, CDll 7 and HLA-DR; and selecting for thosecells that are positive for said at least one of said cell surfacemarkers.
 8. A method according to claim 6 , wherein said reagent is anantibody or an antibody mixture.
 9. A method according to claim 8 ,wherein at least one of said antibodies is fluorochrome conjugated. 10.A method according to claim 9 , wherein said selecting with saidfluorochrome conjugated antibodies is by flow cytometry.
 11. A methodaccording to claim 8 , wherein at least one of said antibodies isconjugated to magnetic particles.
 12. A method according to claim 11 ,wherein said selecting with said magnetic particle conjugated antibodiesis by high gradient magnetic selection.
 13. A substantially purepopulation of hematopoietic progenitor cells, wherein said cells arebound to a reagent that specifically binds to the hematopoieticprogenitor cell antigen recognized by the antibody produced by thehybridoma cell line ATCC HB12346.
 14. A substantially pure population ofhematopoietic progenitor cells according to claim 13 , wherein saidreagent is a monoclonal antibody.
 15. A substantially pure population ofhematopoietic progenitor cells according to claim 14 , wherein saidmonoclonal antibody is produced by the hybridoma cell line ATCC HB12346.16. A substantially pure population of hematopoietic progenitor cellsaccording to claim 15 , wherein said progenitor cells are obtained fromhuman fetal liver.
 17. A substantially pure population of hematopoieticprogenitor cells according to claim 15 , wherein said progenitor cellsare obtained from human peripheral blood.
 18. A substantially purepopulation of hematopoietic progenitor cells according to claim 15 ,wherein said progenitor cells are obtained from human bone marrow.
 19. Asubstantially pure population of hematopoietic progenitor cellsaccording to claim 18 , wherein said bone marrow is adult.
 20. Asubstantially pure population of hematopoietic progenitor cellsaccording to claim 18 , wherein said bone marrow is fetal.
 21. Anisolated nucleic acid molecule, wherein said molecule comprises: (1) afirst sequence having an amino acid coding region for AC133 as set forthin FIG. 12 (SEQ ID NO:1); (2) a second sequence, wherein said secondsequence is a subsequence of said first sequence and is at least 14nucleotides in length; (3) a third sequence in which at least onenucleotide of said first or second sequences is replaced by a differentnucleotide; or (4) a fourth sequence complementary to any of said first,second or third sequences; with the proviso that (i) if said molecule isan RNA molecule, U replaces T in said sequence of said molecule, (ii)said third sequence is at least 90% identical to said first or secondsequence, and (iii) said second sequence is not nucleotides 347-667,1564-1696, or 2010-2386 of SEQ ID NO:1.
 22. The isolated molecule ofclaim 21 , wherein said molecule comprises said first sequence.
 23. Theisolated molecule of claim 21 , wherein said molecule comprises saidsecond sequence.
 24. The isolated molecule of claim 21 , wherein saidmolecule comprises said third sequence.
 25. The isolated molecule ofclaim 21 , wherein said molecule consists essentially of DNA encodingthe amino acid sequence of AC133.
 26. An expression vector comprising anucleic acid sequence of claim 21 .
 27. A cell transfected with themolecule of claim 26 .
 28. An isolated polypeptide, wherein saidpolypeptide comprises: (1) a first amino acid sequence of AC133 as setforth in SEQ ID NO: 2; (2) a second amino acid sequence wherein saidsecond sequence is a subsequence of said first sequences and is at least6 amino acids in length; or (3) a third sequence in which at least oneamino acid of said first or second sequences is replaced by a differentamino acid, with the proviso that said amino acid replacement is areplacement of one acidic residue for another, one basic residue foranother, one non-polar residue for another, one uncharged polar residuefor another, or one aromatic residue for another, with the proviso thatsaid third sequence is at least 90% identical to said first or secondsequence.
 29. The isolated polypeptide of claim 28 , wherein saidpolypeptide comprises said first sequence.
 30. The isolated polypeptideof claim 28 , wherein said polypeptide comprises said second sequence.31. The isolated polypeptide of claim 28 , wherein said polypeptidecomprises said third sequence.
 32. The polypeptide of claim 29 complexedto a ligand.
 33. The polypeptide complex of claim 32 , wherein saidligand is an antibody.
 34. An isolated polypeptide, wherein saidpolypeptide comprises the amino acid sequence from extracellularN-terminus, aa 20-107; first transmembrane region, aa 107-126; firstcytoplasmic loop, aa 127-157; second transmembrane region, aa 158-179;first extracellular loop, aa 180-435; third transmembrane region, aa436-454; second cytoplasmic loop, aa 455-480; fourth transmembraneregion, aa 481-503; second extracellular loop, aa 504-792; fifthtransmembrane, aa 793-816; or cytoplasmic C-terminus, aa 817-865; of SEQID NO:2.
 35. A method for identifying a ligand that binds to humanhematopoetic stem cells, comprising detecting binding of said ligandwith the polypeptide of claim 8 ,.
 36. A reagent that specifically bindsto the polypeptide of claim 28 .
 37. The reagent of claim 36 , whereinsaid reagent is selected from the group consisting of monoclonal andpolyclonal antibodies.
 38. The reagent of claim 36 , wherein saidreagent is a physiological or synthetic ligand.
 39. The polypeptide ofclaim 28 , wherein said polypeptide in not glycosylated.
 40. Thepolypeptide of claim 28 , wherein said polypeptide is glycosylated. 41.In a method of isolating hematopoietic stem cells using a cellseparation technique based on identification of stem cells by selectivebinding of a ligand to an antigenic marker on said stem cell, animprovement which comprises: utilizing an AC133 antigen as saidantigenic marker.
 42. The method of claim 41 , wherein said ligand is anantibody.
 43. The method of claim 41 , wherein said ligand binds to anextracellular region of said AC133 antigen.
 44. The method of claim 41 ,wherein said extracellular region comprises an amino acid selected fromextracellular N-terminus, aa 20-107; first extracellular loop, aa180-435; or second extracellular loop, aa 504-792; of SEQ ID NO:2. 45.The method of claim 41 , wherein said ligand has been identified bydetermining whether compounds in a group of test compounds bind to saidAC133 antigen and selecting said ligand from among compounds that bindspecifically to said AC133 antigen with less than 10% crossreactivitywith any antigen present on mature blood cells.
 46. The method of claim41 , wherein crossreactivity is measured by a competitive binding assaybetween pure AC133 antigen, said ligand, and said suspectedcrossreactive antigen using concentrations of AC133 antigen and saidligand where said ligand half-saturates binding to AC133.
 47. The methodof claim 41 , wherein crossreactivity is measured at a concentration ofAC133 antigen that half saturates monoclonal antibody ATCC HB 12346 whensaid antibody is present at a concentration of 50 ng/100 μl.
 48. Aligand for AC133 identified by the method of claim 36 .
 49. A reagentthat bind specifically to AC133 antigen with less than 5%crossreactivity with any antigen present on mature blood cells.
 50. Thereagent of claim 49 , wherein said reagent is attached to a surface orto a detectible label.
 51. The reagent of claim 49 , wherein said labelis a fluorescent label.